Polypeptide conjugates for intracellular delivery of stapled peptides

ABSTRACT

The present disclosure provides novel polypeptide conjugates. The polypeptide conjugates disclosed herein comprise a stapled peptide comprising a peptide and at least one staple which holds the peptide in an α-helical conformation, and a cyclic cell-penetrating peptide (cCPP) conjugated, directly or indirectly, to the stapled peptide. The present disclosure demonstrates that cCPPs can be used to confer consistent cell-permeability to stapled peptides.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national stage application filed under 35 U.S.C. §371 of PCT/US2018/057894 filed Oct. 28, 2018, which claims the benefitof priority to U.S. Provisional Application No. 62/578,213, filed Oct.27, 2017, the entire contents of which are herein incorporated byreference in its entirety for all purposes.

STATEMENT CONCERNING GOVERNMENT FUNDING

This invention was made with government support under grant nos.R01-GM110208 and R35-GM122459, each awarded by the National Institute ofGeneral Medical Sciences (NIGMS), NIH. The government has certain rightsin the invention.

BACKGROUND

Stapled peptides have emerged as an exciting class of therapeutic agentsfor targeting intracellular protein-protein interactions (PPIs), whichhave been challenging targets for conventional small molecules andbiologics. Verdine G. L., et al., Methods Enzymol. 503, 3-33 (2012);Walensky, L. D., et al., J Med. Chem. 57, 6275-6288 (2014). Theyrecapitulate the structure and specificity of bioactive α-helices,resist proteolytic degradation in vivo, and, when appropriatelydesigned, gain access to the cytosol and nucleus of mammalian cells. Thefirst cellular application of hydrocarbon-stapled α-helices, which weremodeled after the BCL-2 homology 3 (BH3) domain of the pro-apoptoticprotein BID, revealed their capacity for cellular uptake by anenergy-dependent macro-pinocytic mechanism, resulting in activation ofthe apoptotic signaling cascade. Chu, Q., et al., Med. Chem. Commun. 6,111-119 (2015) (clinicaltrials.gov identifier: NCT02264613).

Despite the remarkable promise of stapled peptides as a novel class oftherapeutics for targeting previously intractable proteins, designingstapled peptides with consistent cell-permeability remains a majorchallenge. Many factors including α-helicity, positive charge, peptidesequence, and staple composition and placement appear to affect celluptake propensity. Recently, comprehensive analyses of several hundredstapled peptides in the Verdine and Walensky labs suggest that anoptimal hydrophobic, positive charge, and helical content and properstaple placement are the key drivers of cellular uptake, whereas excesshydrophobicity and positive charge can trigger membrane lysis atelevated peptide dosing. See Chu, Q., et al., Med. Chem. Commun. 6,111-119 (2015); Nature Chemical Biology. 12, 845-852 (2016). It is clearfrom these studies that many stapled peptides are either impermeable orpoorly permeable to the cell membrane, which limits the application ofstapled peptides as therapeutic agents.

Thus, there is a need in the art for improved stapled peptides havingenhanced cellular permeability.

SUMMARY

The instant disclosure provides polypeptide conjugates for intracellulardelivery of stapled peptides. The instant disclosure demonstrates thatcyclic cell-penetrating peptides (cCPPs) can be used to conferconsistent cell-permeability to stapled peptides. In addition, twomethods to staple and conjugate alpha-helical peptides to cCPPs areprovided.

In embodiments, the present disclosure provides for polypeptideconjugates comprising: a stapled peptide comprising a peptide and atleast one staple which holds the peptide in an α-helical conformation,and at least one cyclic cell-penetrating peptide (cCPP) conjugated,directly or indirectly, to the stapled peptide. In embodiments, the cCPPof the present disclosure is conjugated directly or indirectly, to thestaple. In further embodiments, the cCPP is conjugated, directly orindirectly, to the peptide. In still further embodiments, the cCPP isconjugated, directly or indirectly, to the N-terminus of the peptide. Inother embodiments, the cCPP is conjugated, directly or indirectly, tothe C-terminus of the peptide. In further embodiments, the cCPP isconjugated, directly or indirectly, to a side chain of an amino acid ofthe peptide. In the polypeptide conjugates of the instant invention, thestaple may be selected from the group consisting of an amide, alkylene,N-alkylene, alkenylene, alkynylene, aryl, cycloalkyl, cycloalkenyl,cycloalkynyl, heterocyclyl, and heteroaryl, each of which are optionallysubstituted.

The polypeptide conjugates of the instant invention may further comprisea linker, which is covalently bound to an amino acid on the cCPP andeither an amino acid on the peptide or the staple. In some embodiments,the linker is covalently bound to the stapled peptide through adisulfide bond. In further embodiments, the linker may be selected fromthe group consisting of at least one amino acid, alkylene, alkenylene,alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,heteroaryl, ether, and combinations thereof, each of which areoptionally substituted. In embodiments, the linker is capable ofreleasing the stapled peptide from the cCPP after the polypeptideconjugate enters the cytosol of a cell.

The polypeptide conjugates of the instant invention may have a structureaccording to Formula IA, IB, or IC:

wherein:

-   -   each of X and Z, at each instance, are independently selected        from an amino acid;    -   U, at each instance and when present, is independently selected        from an amino acid;    -   J, at each instance and when present, is independently selected        from an amino acid;    -   Z′, at each instance and when present, is independent selected        from an amino acid;    -   a is a number in the range of from 0 to 500;    -   c is at least 3;    -   d is a number in the range of from 1 to 500;    -   e is a number in the range of from 0 to 500;    -   each of g and h are independently and at each instance 0 or 1,        provided in at least one instance g is 1;    -   i is a number in the range of from 0 to 100;    -   Y₁ is an amino acid which has a side chain which forms a first        bonding group (b₁) to the staple, and Y₂ is an amino acid which        has a side chain which forms a second bonding group (b₂) to the        staple.

In some embodiments, c is a number in the range of from 3 to 30. In someembodiments, c is 3, 6, or 10. In further embodiments, each of b₁ and b₂are independently selected from a bond, aryl, thioether, disulfide,amide, ester, and ether.

In embodiments, J is absent, and Z may be either the N-terminus or theC-terminus of the peptide. In embodiments, J is present, e is 1, and Jmay be either the N-terminus or the C-terminus of the peptide. Infurther embodiments, J is present, e is 2 or more, and the terminal J iseither the N-terminus or the C-terminus of the peptide. In otherembodiments, U is absent, and Z′ is either the N-terminus or theC-terminus of the peptide. In embodiments, U is present, a is 1, and Uis either the N-terminus or the C-terminus of the peptide. Inembodiments, U is present, a is 2 or more, and the terminal U is eitherthe N-terminus or the C-terminus of the peptide.

In embodiments, the polypeptide conjugate of Formula IB may have thefollowing structure:

In embodiments, the polypeptide conjugate of Formula IC may have thefollowing structure:

In embodiments, the cCPP may have a sequence comprising Formula II:

wherein:

-   -   each of AA₁, AA₂, AA₃, and AA₄, are independently selected from        a D or L amino acid,    -   each of AA_(u) and AA_(z), at each instance and when present,        are independently selected    -   from a D or L amino acid, and    -   m and n are independently selected from a number from 0 to 6;        and        wherein:    -   at least two amino acids selected from the group consisting of        AA_(u), at each instance and    -   when present, AA₁, AA₂, AA₃, AA₄, and AA_(z), at each instance        and when present, are independently arginine, and    -   at least two of amino acids selected from the group consisting        AA_(u), at each instance and when present, AA₁, AA₂, AA₃, AA₄,        and AA_(z), at each instance and when present, are independently        a hydrophobic amino acid.

In some embodiments, the cCPP has a sequence comprising any of FormulaIIIA-D:

wherein:

-   -   each of AA_(H1) and AA_(H2) are independently a D or L        hydrophobic amino acid;    -   at each instance and when present, each of AA_(u) and AA_(z) are        independently a D or L amino acid; and    -   m and n are independently selected from a number from 0 to 6.

The present disclosure also provides for a cell comprising thepolypeptide conjugates disclosed herein.

The present disclosure additionally provides for a method for cellulardelivery of a stapled peptide, the method comprising contacting a cellwith the polypeptide conjugates disclosed herein.

Further, the present disclosure provides for a method for treating apatient in need thereof, comprising administering the polypeptideconjugates disclosed herein to the patient. The patient may have adisease or condition selected from a cancer, an inflammatory disease orcondition, and an autoimmune disease or condition.

Additionally, the present disclosure provides for a method for makingthe polypeptide conjugates disclosed herein, the method comprisingconjugating a stapled peptide and a cCPP. In other embodiments, thepresent disclosure provides for a method for making a polypeptideconjugates disclosed herein, the method comprising conjugating a peptideto at least one cCPP, and stapling the peptide.

The present disclosure also provides for a pharmaceutical compositioncomprising the polypeptide conjugates disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing a strategy for synthesizing cCPP-stapledpeptide conjugates with DCA as the staple.

FIG. 2 is a survival curve, showing the effect of stapled peptide 2(3-1-1-DCA), CPP9-stapled peptide conjugates 4 and 5 (3-1-1-DCA-CP9peak1 and peak2), and nutlin-3 on the viability of wild type p53 cellline (HCT116 WT) and the p53 knockout cell line (HCT116 p53−/−).

FIGS. 3A-3B is a schematic showing two different strategies forsynthesizing cCPP-stapled peptide conjugates with BBA as thestaple/linker. FIG. 3A shows an on-resin stapling strategy, during whichthe helical peptide, the BBA staple/linker, and the cCPP aresequentially synthesized on resin. FIG. 3B shows a solution-phasestapling strategy, during which the BBA-derivatized cCPP and the helicalpeptide are synthesized separately and then stapled/conjugated in thesolution phase.

FIG. 4 shows a comparison of the cellular entry efficiency of stapledpeptides with and without CPP9 conjugation. HeLa cells were treated with5 μM FITC-labeled peptide for 2 h at 37° C., washed to remove excesspeptide, and subjected to live-cell confocal microscopy. I, DIC; II, GFPchannel; and III, overlap of I and II.

FIG. 5 shows the chemical structure of unstapled peptides 4 and 5(stereoisomers), an HPLC chromatogram, and a low-resolution MALDI-TOF MSspectrum for the product (retention time=32 minutes).

FIG. 6 shows the chemical structure of stapled peptide 3, an HPLCchromatogram, and a low-resolution MALDI-TOF MS spectrum for the product(retention time=30.5 minutes).

FIG. 7 shows the chemical structure of aminoxy-cCPP9, an HPLCchromatogram, and a low-resolution MALDI-TOF MS spectrum for aminoxycCPP9 (retention time=22 min).

FIG. 8A shows the chemical structure of stapled peptide 4 and 5(stereoisomers) which has been conjugated to a cCPP (cCPP 9) via alinker and an HPLC chromatogram. FIG. 8B shows low-resolution MALDI-TOFMS spectra for aminoxy-cCPP9 (peak at retention time=23 minutes),structure 4 (peak at retention time=33.5 minutes), and structure 5 (peakat retention time=34.5 minutes).

FIG. 9 shows the chemical structure for stapled, labeled peptide 10,HPLC chromatograms and an MS spectrum.

FIGS. 10A-10B shows the chemical structure of stapled, labeled peptide11 conjugated to a cCPP via a linker, HPLC chromatograms (FIG. 10A) andan MS spectrum (FIG. 10B).

FIGS. 11A-11B shows the chemical structure of stapled, labeled peptide12, HPLC chromatograms (FIG. 11A) and MS spectra (FIG. 11B).

FIGS. 12A-12B shows the chemical structure of stapled, labeled peptide13 conjugated to a cCPP via a linker, HPLC chromatograms (FIG. 12A), andan MS spectrum (FIG. 12B).

FIG. 13 shows the chemical structure of stapled, labeled peptide 14,HPLC chromatograms, and an MS spectrum.

FIGS. 14A-14B shows the chemical structure of stapled, labeled peptide15 conjugated to a cCPP via a linker, HPLC chromatograms (FIG. 14A) anda MS spectrum (FIG. 14B).

FIG. 15 shows the chemical structure of a stapled, labeled peptide 16,HPLC chromatograms, and a MS spectrum.

FIGS. 16A-16B shows the chemical structure of a stapled, labeled peptide17 conjugated to a cCPP via a linker, HPLC chromatograms (FIG. 16A) andan MS spectrum (FIG. 16B).

FIG. 17 shows the chemical structure of a stapled, labeled peptide 18,HPLC chromatograms, and a MS spectrum.

FIGS. 18A-18B shows the chemical structure of a stapled, labeled peptide19 conjugated to a cCPP via a linker, HPLC chromatograms (FIG. 18A) anda MS spectrum (FIG. 18B).

FIG. 19 shows the chemical structure of a stapled, labeled peptide 21,which has been conjugated to a cCPP via a linker, and HPLCchromatograms.

FIGS. 20A-20D shows the chemical structures of amide stapled peptidesand conjugates, including sPDI peptide 22 (FIG. 20A), CPP9-sPDI peptideconjugate 23 (FIG. 20B), R9-sPDI peptide conjugate 24 (FIG. 20C), andTat-sPDI peptide conjugate 25 (FIG. 20D). CPP9, R9, and Tat are eachconjugated to the peptide via a linker attached to the C-terminus.

FIG. 21 shows a comparison of the cellular entry efficiency of stapledpeptides with and without conjugation. Images are provided fromstructure 22 (sPDI), structure 23 (CPP9-sPDI), structure 24 (R9-sPDI),and structure 25 (Tat-sPDI). Analogs with Lys (FITC) at the N-terminuswere used for confocal imaging.

FIG. 22 shows a graph for a cell free competition assay comparing thefunctional cytosolic delivery of sPDI (structure 22), CPP9-sPDI(structure 23), and CPP9-sPDI F10A mutant. The data for the fluorescencepolarization (FP) plot was obtained using FITC-labeled MDM2 ligand (15nM) in the presence of MDM2 (15 nM) and unlabeled sPDI, CPP9-conjugatedstapled PDI (CPP9-sPDI; structure 23), or CPP9-sPDI F, 10A mutant (0-5μM) as a function of competitor peptide concentration.

FIG. 23 shows a graph for an anti-proliferation assay comparing theeffect of 72-hour treatment with CPP9-sPDI (structure 23), Nutlin-3a,R9-sPDI (structure 24), sPDI (structure 22), CPP9-sPDI(F10A) andTat-sPDI (structure 25, 0-20 μM) on the viability of SJSA-1 cell line inthe presence of 10% FBS as measured by MTT assay. IC₅₀ values (μM) areprovided for each test compound.

FIG. 24 is a graph showing that the anti-proliferative activity ofCPP9-sPDI (structure 23) is mediated by apoptotic pathways. Thepercentage of Annexin V+/PI+ and Annexin V+/PI− SJSA-1 cells after48-hour treatment of inhibitors in presence of 10% FBS was measured.

FIG. 25 is a graph showing the stability of CPP9-sPDI (structure 23) in25% human serum at 37° C.

DETAILED DESCRIPTION

When describing the present invention, all terms not defined herein havetheir common art-recognized meanings. Any term or expression notexpressly defined herein shall have its commonly accepted definitionunderstood by those skilled in the art. To the extent that the followingdescription is of a specific embodiment or a particular use of theinvention, it is intended to be illustrative only, and not limiting ofthe claimed invention. The following description is intended to coverall alternatives, modifications and equivalents that are included in thespirit and scope of the invention, as defined in the appended claims.

Definitions

“Amino acid” as used herein refers to the moiety that is present in thestapled peptide conjugates of the present disclosure. As used herein“hydrophobic amino acid” refers to an amino acid that has a hydrophobicgroup (e.g., an alkyl chain) on the side chain. Similarly, an “aromaticamino acid” refers to an amino acid having an aromatic group (e.g., aphenyl) on the side chain.

“Alkylene” or “alkylene chain” refers to a fully saturated, straight orbranched divalent hydrocarbon chain radical, having from one to fortycarbon atoms. Non-limiting examples of C₂-C₄₀ alkylene include ethylene,propylene, n-butylene, ethenylene, propenylene, n-butenylene,propynylene, n-butynylene, and the like. In some embodiments, thealkylene chain is attached, directly or indirectly, to the cCPP througha single bond and, directly or indirectly, to the staple or the peptidethrough a single bond. In some embodiments, the alkylene chain isindependently attached, directly or indirectly, to side chain of a firstamino acid of the peptide and a second amino acid of a peptide. Unlessstated otherwise specifically in the specification, an alkylene chaincan be optionally substituted as described herein.

“Alkenylene” or “alkenylene chain” refers to a straight or brancheddivalent hydrocarbon chain radical, having from two to forty carbonatoms, and having one or more carbon-carbon double bonds. Non-limitingexamples of C₂-C₄₀ alkenylene include ethene, propene, butene, and thelike. In some embodiments, the alkenylene chain is attached, directly orindirectly, to the cCPP through a single bond and, directly orindirectly, to the staple or the peptide through a single bond. In someembodiments, the alkenylene chain is independently attached, directly orindirectly, to side chain of a first amino acid of the peptide and asecond amino acid of a peptide. Unless stated otherwise specifically inthe specification, an alkenylene chain can be optionally substituted.

“Alkynylene” or “alkynylene chain” refers to a straight or brancheddivalent hydrocarbon chain radical, having from two to forty carbonatoms, and having one or more carbon-carbon triple bonds. Non-limitingexamples of C₂-C₄₀ alkynylene include ethynylene, propargylene and thelike. In some embodiments, the alkynylene chain is attached, directly orindirectly, to the cCPP through a single bond and, directly orindirectly, to the staple or the peptide through a single bond. In someembodiments, the alkynylene chain is independently attached, directly orindirectly, to side chain of a first amino acid of the peptide and,directly or indirectly, to a second amino acid of a peptide. Unlessstated otherwise specifically in the specification, an alkynylene chaincan be optionally substituted.

“Aryl” refers to a hydrocarbon ring system divalent radical comprisinghydrogen, 6 to 40 carbon atoms and at least one aromatic ring. Forpurposes of this invention, the aryl divalent radical can be amonocyclic, bicyclic, tricyclic or tetracyclic ring system, which caninclude fused or bridged ring systems. Aryl divalent radicals include,but are not limited to, aryl divalent radicals derived fromaceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene,benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene,indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene,and triphenylene. In some embodiments, the aryl divalent radical isattached, directly or indirectly, to the cCPP through a single bond and,directly or indirectly, to the staple or the peptide through a singlebond. In some embodiments, the aryl is independently attached, directlyor indirectly, to side chain of a first amino acid of the peptide and,directly or indirectly, to either the staple or a second amino acid of apeptide. Unless stated otherwise specifically in the specification, anaryl group can be optionally substituted.

“Cycloalkyl” refers to a stable non-aromatic monocyclic or polycyclicfully saturated hydrocarbon divalent radical having from 3 to 40 carbonatoms and at least one ring, wherein the ring consists solely of carbonand hydrogen atoms, which can include fused or bridged ring systems.Monocyclic cycloalkyl divalent radicals include, for example,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl. Polycyclic cycloalkyl divalent radicals include, forexample, adamantyl, norbornyl, decalinyl,7,7-dimethyl-bicyclo[2.2.1]heptanyl, and the like. In some embodiments,the cycloalkyl divalent radical is attached, directly or indirectly, tothe cCPP through a single bond and, directly or indirectly, to thestaple or the peptide through a single bond. In some embodiments, thecycloalkyl is independently attached, directly or indirectly, to sidechain of a first amino acid of the peptide and, directly or indirectly,to either the staple or a second amino acid of a peptide. Unlessotherwise stated specifically in the specification, a cycloalkyl groupcan be optionally substituted.

“Cycloalkenyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon divalent radical having from 3 to 40 carbon atoms, at leastone ring having, and one or more carbon-carbon double bonds, wherein thering consists solely of carbon and hydrogen atoms, which can includefused or bridged ring systems. Monocyclic cycloalkenyl radicals include,for example, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like. Polycyclic cycloalkenyl radicals include, for example,bicyclo[2.2.1]hept-2-enyl and the like. In some embodiments, thecycloalkenyl divalent radical is attached, directly or indirectly, tothe cCPP through a single bond and, directly or indirectly, to thestaple or the peptide through a single bond. In some embodiments, thecycloalkenyl is independently attached, directly or indirectly, to sidechain of a first amino acid of the peptide and, directly or indirectly,to either the staple or a second amino acid of a peptide. Unlessotherwise stated specifically in the specification, a cycloalkenyl groupcan be optionally substituted.

“Cycloalkynyl” refers to a stable non-aromatic monocyclic or polycyclichydrocarbon divalent radical having from 3 to 40 carbon atoms, at leastone ring having, and one or more carbon-carbon triple bonds, wherein thering consists solely of carbon and hydrogen atoms, which can includefused or bridged ring systems. Monocyclic cycloalkynyl radicals include,for example, cycloheptynyl, cyclooctynyl, and the like. In someembodiments, the cycloalkynyl divalent radical is attached, directly orindirectly, to the cCPP through a single bond and, directly orindirectly, to the staple or the peptide through a single bond. In someembodiments, the cycloalkynyl is independently attached, directly orindirectly, to side chain of a first amino acid of the peptide and,directly or indirectly, to either the staple or a second amino acid of apeptide. Unless otherwise stated specifically in the specification, acycloalkynyl group can be optionally substituted.

“Heterocyclyl,” “heterocyclic ring” or “heterocycle” refers to a stable3- to 20-membered aromatic or non-aromatic ring divalent radical whichconsists of two to twelve carbon atoms and from one to six heteroatomsselected from the group consisting of nitrogen, oxygen and sulfur.Heterocyclycl or heterocyclic rings include heteroaryls as definedbelow. Unless stated otherwise specifically in the specification, theheterocyclyl radical can be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which can include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical can be optionally oxidized; the nitrogen atom can be optionallyquaternized; and the heterocyclyl radical can be partially or fullysaturated. Examples of such heterocyclyl radicals include, but are notlimited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl,imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl,morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl,2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl,piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl,thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl,thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and1,1-dioxo-thiomorpholinyl. In some embodiments, the heterocyclyldivalent radical is attached, directly or indirectly, to the cCPPthrough a single bond and, directly or indirectly, to the staple or thepeptide through a single bond. In some embodiments, the heterocyclyl isindependently attached, directly or indirectly, to side chain of a firstamino acid of the peptide and, directly or indirectly, to either thestaple or a second amino acid of a peptide. Unless stated otherwisespecifically in the specification, a heterocyclyl group can beoptionally substituted.

“Heteroaryl” refers to a 5- to 20-membered ring system radicalcomprising hydrogen atoms, one to thirteen carbon atoms, one to sixheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur, and at least one aromatic ring. For purposes of this invention,the heteroaryl radical can be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which can include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heteroarylradical can be optionally oxidized; the nitrogen atom can be optionallyquaternized. Examples include, but are not limited to, azepinyl,acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl,benzofuranyl, benzooxazolyl, benzothiazolyl, benzothiadiazolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl(benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl,carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl,furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl,isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl,isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl,1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl,phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl,pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl,quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl,tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl,triazinyl, and thiophenyl (i.e. thienyl). In some embodiments, theheteroaryl divalent radical is attached, directly or indirectly, to thecCPP through a single bond and, directly or indirectly, to the staple orthe peptide through a single bond. In some embodiments, the heteroarylis independently attached, directly or indirectly, to side chain of afirst amino acid of the peptide and, directly or indirectly, to eitherthe staple or a second amino acid of a peptide. Unless stated otherwisespecifically in the specification, a heteroaryl group can be optionallysubstituted.

The term “ether” used herein refers to a divalent radical moiety havinga formula—[(R₁)_(m)—O—(R₂)_(n)]_(z)— wherein each of m, n, and z areindependently selected from 1 to 40, and each of R₁ and R₂ areindependently an alkylene, alkenylene, alkynylene, aryl, heteroaryl,cycloalkyl, cycloalkenyl, cycloalkynyl, or heterocyclyl group. In someembodiments, each of R₁ and R₂ are independently straight or branchedalkylene groups. In particular embodiments, the ether has theformula—[(CH₂)_(m)—O—(CH₂)_(n)]_(z)— wherein each of m, n, and z areindependently selected from 1 to 40. Examples include polyethyleneglycol. The ether is attached, directly or indirectly, to the cCPPthrough a single bond and, directly or indirectly, to the staple or thepeptide through a single bond. Unless stated otherwise specifically inthe specification, the ether can be optionally substituted.

The term “N-alkylene” used herein refers to an alkylene divalent radicalas defined above containing at least one nitrogen atom and where a pointof attachment of the alkylene radical to the rest of the molecule isthrough the alkylene radical. In some embodiments, the point ofattachment may optionally be the nitrogen atom. Unless stated otherwisespecifically in the specification, a N-alkylene group can be optionallysubstituted.

As used herein, a “peptide” or “polypeptide” comprises a polymer ofamino acid residues linked together by peptide (amide) bonds. Theterm(s), as used herein, refer to proteins, polypeptides, and peptide ofany size, structure, or function. Typically, a peptide or polypeptidewill be at least three amino acids long. A peptide or polypeptide mayrefer to an individual protein or a collection of proteins. The peptidesof the instant invention may contain natural amino acids and/ornon-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain). Amino acid analogsas are known in the art may alternatively be employed. One or more ofthe amino acids in a peptide or polypeptide may be modified, forexample, by the addition of a chemical entity such as a carbohydrategroup, a hydroxyl group, a phosphate group, a farnesyl group, anisofarnesyl group, a fatty acid group, a linker for conjugation,functionalization, or other modification. A peptide or polypeptide mayalso be a single molecule or may be a multi-molecular complex, such as aprotein. A peptide or polypeptide may be just a fragment of a naturallyoccurring protein or peptide. A peptide or polypeptide may be naturallyoccurring, recombinant, or synthetic, or any combination thereof.

“Stapling” or “peptide stapling” is a strategy for constraining peptidestypically in an alpha-helical conformation. Stapling is carried out bycovalently linking the side-chains of two amino acids on a peptide,thereby forming a peptide macrocycle. Stapling generally involvesintroducing into a peptide at least two moieties capable of undergoingreaction to generate at least one cross-linker between the at least twomoieties. The moieties may be two amino acids with appropriate sidechains that are introduced into peptide sequence or the moieties mayrefer to chemical modifications of side chains. Stapling provides aconstraint on a secondary structure, such as an alpha-helical structure.The length and geometry of the cross-linker can be optimized to improvethe yield of the desired secondary structure content. The constraintprovided can, for example, prevent the secondary structure fromunfolding and/or can reinforce the shape of the secondary structure. Asecondary structure that is prevented from unfolding is, for example,more stable.

A “stapled peptide” is a peptide comprising a staple (as described indetail herein). More specifically, a stapled peptide is a peptide inwhich one or more amino acids on the peptide are cross-linked to holdthe peptide in a particular secondary structure, such as analpha-helical conformation. The peptide of a stapled peptide comprises aselected number of natural or non-natural amino acids, and furthercomprises at least two moieties which undergo a reaction to generate atleast one cross-linker between the at least two moieties, whichmodulates, for example, peptide stability.

A “stitched” peptide, is a stapled peptide comprising more than one(e.g., two, three, four, five, six, etc.) staple.

The term “substituted” used herein means any of the above groups (i.e.,alkylene, alkenylene, alkynylene, aryl, carbocyclyl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, and/or ether)wherein at least one hydrogen atom is replaced by at least onenon-hydrogen atom such as, but not limited to: a halogen atom such as F,Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxygroups, and ester groups; a sulfur atom in groups such as thiol groups,thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups;a nitrogen atom in groups such as amines, amides, alkylamines,dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, andtriarylsilyl groups; and other heteroatoms in various other groups.“Substituted” also means any of the above groups in which one or morehydrogen atoms are replaced by a higher-order bond (e.g., a double- ortriple-bond) to a heteroatom such as oxygen in oxo, carbonyl, carboxyl,and ester groups; and nitrogen in groups such as imines, oximes,hydrazones, and nitriles. For example, “substituted” includes any of theabove groups in which one or more hydrogen atoms are replaced with—NR_(g)R_(h), —NR_(g)C(═O)R_(h), —NR_(g)C(═O)NR_(g)R_(h),—NR_(g)C(═O)OR_(h), —NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g),—SR_(g), —SOR_(g), —SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and—SO₂NR_(g)R_(h). “Substituted also means any of the above groups inwhich one or more hydrogen atoms are replaced with —C(═O)R_(g),—C(═O)OR_(g), —C(═O)NR_(g)R_(h), —CH₂SO₂R_(g), —CH₂SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino,thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl,cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. “Substituted” further means any of the above groups inwhich one or more hydrogen atoms are replaced by a bond to an amino,cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkenyl,alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl,haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl,heteroaryl, N-heteroaryl and/or heteroarylalkyl group. In addition, eachof the foregoing substituents can also be optionally substituted withone or more of the above substituents. Further, those skilled in the artwill recognize that “substituted” also encompasses instances in whichone or more hydrogen atoms on any of the groups described herein arereplaced by a functional group, and the functional group undergoes areaction to form a covalent bond with the cCPP, the staple or thepeptide. The reaction product is also considered a substituent. Forexample, in embodiments where the linker is conjugated to the staple,the staple may be appropriately substituted with a group that is capableof forming a bond to the linker. In some embodiments, said sample may besubstituted with a carbonyl group (e.g., ketone or aldehyde), whichforms an oxime upon coupling with the linker having a nucleophilichydroxylamine (e.g., FIG. 1). In another example, any of the abovegroups can be substituted at a first position with a carboxylic acid(i.e., —C(═O)OH) which forms an amide bond with an appropriate aminoacid CPP (e.g., lysine). Alternatively, or in addition, any of the abovegroups can be substituted with either an electrophilic group (e.g.,—C(═O)H, —CO₂R_(g), -halide, etc. where R_(g) is a leaving group) whichforms a bond with the N-terminus of the peptide or a nucleophilic group(—NH₂, —NHR_(g), —OH, etc.) which forms a bond with the C-terminus ofthe peptide. In other embodiments, the group is substituted with a thiolgroup which forms a disulfide bond with a cysteine (or amino acid analoghaving a thiol group) in the peptide.

The term “radical” as used herein in reference to the above groups referto an electron that participates in forming a bond to the moiety towhich it is attached. For example, when the polypeptide conjugatesdisclosed herein comprise an ether linker which conjugates the cCCP tothe stapled peptide. Prior to conjugation, the ether linker is definedas a divalent radical. To form the polypeptide conjugate one electron ofthe divalent radical is shared in a single bond to the cCCP, and theother electron is shared in a single bond with the stapled peptide.

The term “indirectly” when used in conjunction with attached orconjugated refers to a connection between groups (e.g., a cCPP and astapled peptide), which is achieved using a linker. For example, alinker can be used to indirectly attach a cCPP to a staple, according tosome embodiments.

Polypeptide Conjugates

The present disclosure, in various embodiments, provides for polypeptideconjugates comprising: a stapled peptide comprising a peptide and atleast one staple which holds the peptide in an α-helical conformation,and at least one cyclic cell-penetrating peptide (cCPP) conjugated,directly or indirectly, to the stapled peptide. The cCPP can beconjugated to the stapled peptide at any suitable location. In someembodiments, the cCPP may be conjugated directly or indirectly, to thestaple. In other embodiments, the cCPP may be conjugated, directly orindirectly, to the peptide at any appropriate position, including to aside chain of an amino acid in the peptide or to the N- or C-terminus ofthe peptide. Thus, in some embodiments, the cCPP may be conjugated,directly or indirectly, to the N-terminus of the peptide. In otherembodiments, the cCPP may be conjugated, directly or indirectly, to theC-terminus of the peptide. In still other embodiments, the cCPP may beconjugated, directly or indirectly, to a side chain of an amino acid ofthe peptide.

The polypeptide conjugates of the instant invention may have a structureaccording to Formula IA, IB, or IC:

In some embodiments, each of X and Z, at each instance, areindependently selected from an amino acid. In some embodiments, U, ateach instance and when present, is independently selected from an aminoacid. In some embodiments, J, at each instance and when present, isindependently selected from an amino acid. In some embodiments, Z′, ateach instance and when present, is independent selected from an aminoacid.

In some embodiments, d is a number in the range of from 1 to 500. Insome embodiments, e is a number in the range of from 0 to 500. In someembodiments, i is a number in the range of from 0 to 100.

In some embodiments, each of g and h are independently and at eachinstance 0 or 1, provided in at least instance g is 1. Thus, in someembodiments, the peptide conjugates may comprise 1 cCPP-linker moiety(e.g., when d=1, g=1, and h=0 in Formula IB) or more than cCPP-linkermoiety (e.g., when d=2, g=2, and h=0 in Formula IB; or when d=10, g=2,and h=0 in Formula IB).

In some embodiments, a is a number in the range of from 0 to 500. Insome embodiments, c is at least 3. In some embodiments, c may be anynumber, 3 or greater, such that the staple (as described herein) is thesame face of the alpha helix. In some embodiments, c is 3, 6, or 10. Infurther embodiments, each of b₁ and b₂ are independently selected from abond, aryl, thioether, disulfide, amide, ester, and ether.

In some embodiments, Y₁ is an amino acid which has a side chain whichforms a first bonding group (b₁) to the staple, and Y₂ is an amino acidwhich has a side chain which forms a second bonding group (b₂) to thestaple.

The present disclosure envisions that the structures of Formula IA, IB,or IC can be interpreted as having an N to C or C to N orientation. Thatis, the top of the structure can be either the N-termini or theC-termini. Similarly, the bottom of the structure can be either theC-termini or the N-termini. In embodiments, J is absent, and Z may beeither the N-terminus or the C-terminus of the peptide. In embodiments,J is present, e is 1, and J may be either the N-terminus or theC-terminus of the peptide. In further embodiments, J is present, e is 2or more, and the terminal J is either the N-terminus or the C-terminusof the peptide. In other embodiments, U is absent, and Z′ is either theN-terminus or the C-terminus of the peptide. In embodiments, U ispresent, a is 1, and U is either the N-terminus or the C-terminus of thepeptide. In embodiments, U is present, a is 2 or more, and the terminalU is either the N-terminus or the C-terminus of the peptide.

In embodiments, the polypeptide conjugate of Formula IB may have thefollowing structure:

In embodiments, the polypeptide conjugate of Formula IC may have thefollowing structure:

Peptide

The peptide for use in the polypeptide conjugates disclosed herein maybe any peptide which contain at least one region having alpha-helicalstructure. The alpha-helix is a common secondary structure motif andplays an important functional role in many proteins. In embodiments, thepeptide may be mostly in alpha-helical conformation, or the peptide maybe part of a larger protein that includes one or more alpha-helicalregions. As discussed above, the staple is appropriate located tosubstantially maintain the alpha-helical conformation.

The peptide may be naturally occurring, or it may be specificallydesigned to interact with a target (e.g., to inhibit protein-proteininteractions). In some embodiments, the peptide may be derived from anaturally occurring peptide, which appropriate modifications tofacilitate conjugation with the staple, linker, and/or cCPP, orcombinations thereof. Thus, the amino acids in the peptide (each of X,Z, U, J, Y₁, Y₂, and Z′, at each instance and when present) areindependently selected from any natural or non-natural amino acid, andmay independently refer to amino acids that naturally occur in thepeptide or introduced into a peptide. The term “non-natural amino acid”refers to an organic compound that is analog of a natural amino acid inthat it has a structure similar to a natural amino acid so that itmimics the structure and reactivity of a natural amino acid. Thenon-natural amino acid can be a modified amino acid, and/or amino acidanalog, that is not one of the 20 common naturally occurring amino acidsor the rare natural amino acids selenocysteine or pyrrolysine.Non-natural amino acids can also be the D-isomer of the natural aminoacids. Examples of suitable amino acids include, but are not limited to,alanine, alloisoleucine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, naphthylalanine, phenylalanine, proline,pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine,2,3-diaminopropionic acid a derivative, or combinations thereof. These,and others, are listed in the Table 1 along with their abbreviationsused herein.

TABLE 1 Amino Acid Abbreviations Abbreviations* Abbreviations* AminoAcid L-amino acid D-amino acid Alanine Ala (A) ala (a) AllosoleucineAIle aile Arginine Arg (R) arg (r) Asparagine Asn (N) asn (n) asparticacid Asp (D) asp (d) Cysteine Cys (C) cys (c) Cyclohexylalanine Cha cha2,3-diaminopropionic acid Dap dap 4-fluorophenylalanine Fpa (Σ) fpaglutamic acid Glu (E) glu (e) glutamine Gln (Q) gln (q) glycine Gly (G)gly (g) histidine His (H) his (h) Homoproline (aka pipecolic acid) Pip(Θ) pip (θ) isoleucine Ile (I) ile (i) leucine Leu (L) leu (l) lysineLys (K) lys (k) methionine Met (M) met (m) naphthylalanine Nal (Φ) nal(ϕ) norleucine Nle (Ω) nle (ω) phenylalanine Phe (F) phe (f)phenylglycine Phg (Ψ) Phg (ψ) 4-(phosphonodifluorometh- F₂Pmp (Λ) f₂pmp(λ) yl)phenylalanine proline Pro (P) pro (p) sarcosine Sar (Ξ) sar (ξ)Selenocysteine Sec (U) sec (u) Serine Ser (S) ser (s) Threonine Thr (T)thr (y) Tyrosine Tyr (Y) tyr (y) Tryptophan Trp (W) trp (w) Valine Val(V) val (v) 2,3-diaminopropionic acid Dap dap *single letterabbreviations: when shown in capital letters herein it indicates theL-amino acid form, when shown in lower case herein it indicates theD-amino acid.

In some embodiments, Y₁ is an amino acid which has a side chain thatforms a first bonding group (b₁) to the staple, and Y₂ is an amino acidwhich has a side chain that forms a second bonding group (b₂) to thestaple. Thus, precursors of each of Y₁ and Y₂ may independently be anyamino acid having a side chain which is suitable, or can be modified tobe suitable, to covalently bind the staple. Non-limiting examples ofsuch amino acids include cysteine, glutamine, asparagine, and lysine,and analogs thereof (e.g., having additional hydrocarbons in the sidechain, such as homocysteine).

Further examples of amino acid analogs which can be introduced to thepeptides disclosed herein include those having an alkene side chain, analkyne side chain or a nitrile side chain, as these side chains may beused to form the staple (e.g., during olefin or ring closing metathesisbetween two alkene-containing side chains) or to conjugate the staple.In still other embodiments, the precursor of Y₁ may be an amino acidhaving a side chain which is suitable for covalently bonding (e.g.,forming an amide bond) to a side chain of the precursor of Y₂. In suchembodiments, the “reaction product” between side chains of these aminoacid analogs is the staple. For example, in certain embodiments, theprecursor to Y₁ is lysine and the precursor to Y₂ is aspartate, and theamino group on the side chain of the Y₁ precursor reacts with thecarboxyl group on the side chain of the Y₂ precursor to form an amide,which is the staple. As another example, the precursor to Y₁ may be anamino acid analog having a alkyne on the side chain and the precursor toY₂ may be an amino acid having an azide on the side chain, and thesegroups react to form a triazole.

In particular, embodiments, the peptide can comprise one or more aminoacids having a side chain comprising a thiol group (i.e., prior toconjugation to the linker, cCPP, and/or staple). The thiol group may beused to conjugate the cCPP, linker, and/or staple, by forming thioether,thioester, or disulfide. Non-limiting examples of amino acid analogshaving a thiol group include cysteine, homocysteine, and any of thefollowing amino acid analogs:

As previously stated, the above groups are precursors which allow forconjugation of a staple, linker, and/or a cCCP. Specifically, in orderto conjugate the staple, linker, and/or a cCCP to the peptide, thehydrogen of the thiol in the above group is replaced by a bond to thestaple, linker, or the cCPP.

One example of a peptide for use in the instant invention is a ligand ofthe MDM2 protein, such as the alpha-helical peptide Ac-LTFEHYWAQLTS (SEQID NO: 1) (“PDI”). This ligand is capable of binding to the MDM2 proteinand therefore disrupting the interaction of MDM2 with p53. Peptides thatdisrupt the MDM2/p53 interaction might be useful for many applications,including, but not limited to, control of soft tissue sarcomas (whichoverexpresses MDM2 in the presence of wild type p53). These cancers maybe held in check with small molecules that could intercept MDM2, therebypreventing suppression of p53. Peptides of the instant invention may besynthesized according to methods known to those of skill in the art. Forexample, the peptides may be synthesized using standard solid-phasepeptide synthesis (SPPS).

Staple

The staple described herein stabilizes the bioactive, alpha-helicalstructure of the peptide, conferring, for example, protease resistance,cellular penetrance, and biological activity. The staple may be anysynthetic brace capable of holding the peptide in an alpha-helicalconformation. In embodiments, the staple reinforces the nativealpha-helical conformation of the peptide, thereby maintaining bindingaffinity towards its protein targets.

Methods for peptide stapling are known to those of skill in the art. Insome embodiments, peptide stapling may require generation of apolypeptide comprising two natural or non-natural amino acids (i.e.,precursors of Y₁ and Y₂) bearing side chains with functional groups thatare suitable for stapling. In certain embodiments, the sides of theprecursors of Y₁ and Y₂ can react to form the staple. In otherembodiments, the side precursors of Y₁ and Y₂ have side chains suitablefor conjugating a staple (i.e., side chains with appropriate functionalgroups to bind the staple by forming of bonding groups, b₁ or b₂). Instill other embodiments, the staple is formed by replacing anintramolecular hydrogen bond with a covalent bond, for example byreplacing the hydrogen atom and carbonyl group on the opposing aminoacids that participate in the intramolecular hydrogen bondinginteraction with a group that crosslinks said opposing amino acids.Examples of such modifications are described in Joy, S. T. et al., Chem.Commun (Camb.) 52 (33), 5738-5741), and Zhao, H. et al. Angew. Chem.Int. Ed. 2016, 55, 12088-12093, each of which are herein incorporated byreference in its entirety.

The amino acids which form or are bound to the staple are typicallyspaced apart in the peptide chain such that their side chains are onsubstantially the same face of the folded peptide. Thus, for analpha-helical peptide, the amino acid side chains are typically locatedon substantially the same face of the alpha helix. The distance betweenopposing amino acids on the same face of the peptide per turn of thehelix is about 5.4 Å. Accordingly, in various embodiments, the staple isany appropriate moiety which holds these opposing amino acids at adistance of about 5.4 Å, thereby maintaining the alpha helicalconformation. Thus, in embodiments, the staple may have a size in therange of from about 5 Å to about 6 Å, of from about 10 Å to about 12 Å,of from about 15 Å to about 17 Å, of from about 21 Å to about 23 Å, offrom about 26 Å to about 28 Å, and of from about 31 Å to about 34 Å,inclusive of all values and subranges therebetween. In otherembodiments, the staple may have a size of about 5 Å, about 5.5 Å, about6 Å, about 10.5, about 11 Å, about 11.5 Å, about 12 Å, about 16.5 Å,about 17 Å, about 17.5 Å, about 22 Å, about 22.5 Å, about 23 Å, about23.5 Å, about 25.5 Å, about 26 Å, about 26.5 Å, about 27 Å, about 27.5Å, about 28 Å, about 28.5 Å, about 30.5 Å, about 31 Å, about 31.5 Å,about 32 Å, about 32.5 Å, about 33 Å, about 33.5 Å, about 34 Å, or about34.5 Å.

For single turn stapling in an alpha helix, the amino acids to which thestaple is conjugated are generally located at the i, i+4 positions. Fordouble turn stapling in an alpha helix, the amino acids are generallylocated at the i, i+7 positions. For triple turn stapling in an alphahelix, the amino acids are generally located at the i, i+11 positions.In other embodiments, the polypeptide conjugates disclosed herein cancomprise two or more staples (also referred to as stitched peptides).For example, the staple can be located at the i, i+4 positions and atthe i+7, i+11.

In various embodiments, the number of amino acids between Y₁ andY₂—i.e., “c” in Formula IA-IC—is an appropriate number of amino acidssuch that the staple is located on substantially the same face of thealpha helix. In embodiments, c is at least 3. In other embodiments, c isa number from 3 to 30. In still other embodiments, c is 3, 6, or 10.

In some embodiments, the staple is selected from the group consisting ofalkylene, N-alkylene, alkenylene, alkynylene, aryl, cycloalkyl,cycloalkenyl, cycloalkynyl, heterocyclyl, and heteroaryl, each of whichare optionally substituted. Non-limiting examples of staples include alactam staple, a hydrocarbon staple, a CuAAC staple, a bis-thioetherstaple, a perfluorobenzene staple, and a thioether staple.

A number of alternative stapling methods are known to those in the art,each using a different form of macrocyclization chemistry and givingrise to stapled peptides with different bioactive properties. Forexample, the stapling may be one-component stapling. One-componentstapling involves a direct bond-forming reaction between the side-chainsof two amino acids. In some embodiments, the one-component staplingtechnique may comprise formation of an amide bond between to side chainsof amino acids in the peptide. In some embodiments, the one-componentstapling technique may comprise, for example, a ring-closing metathesis,a lactamization, a cycloaddition (such as the Cu(I)-catalyzedazide-alkyne cycloaddition (CuAAC, “click reaction”)), a reversiblereaction (such as formation of a disulfide bridge or an oxime linkage),or thioether formation. The stapling technique may alternatively betwo-component stapling. Two-component stapling involves a bifunctionallinker compound which forms a staple by reacting with two complementarynative or non-native amino acids in the peptide of interest.Two-component stapling may employ, for example, a photoswitchable linkeror a functionalized “double click” linker. When the staple is conjugatedvia click reaction, each of b₁ and b₂ are a triazole, which may beoptionally substituted. That is, in some embodiments, the precursors toY₁ and Y₂ may independently be an amino acid analog having an alkynegroup on the side chain or an amino acid having an azide group on theside chain, and these groups react with a precursor to the staple havingcomplementary alkyne and/or azide groups to form a triazole. The clickreaction may also be used to produce a staple by two-component stapling,in which case the staple is the triazole and b₁ and b₂ are absent. Thus,b₁ and b₂ may independently be the bonding group formed when any of theabove techniques are used to conjugate to staple to the peptide. In someembodiments, each of b₁ and b₂ are independently absent or selected fromaryl (e.g., triazole), thioether, disulfide, amide, ester, and ether.

Additional examples of staples and stapling methods appropriate for usein the stapled peptides of the instant invention are described inWalensky, L. D., et al., J. Med. Chem., 57, 6275-6288 (2014), Lau, Y.H., et al., Chem. Soc. Rev., 00, 1-12 (2014), Joy, S. T. et al., Chem.Commun (Camb.) 52 (33), 5738-5741), and Zhao, H. et al. Angew. Chem.Int. Ed. 2016, 55, 12088-12093, each of which are incorporated herein byreference in their entireties.

Cyclic Cell-Penetrating Peptide (cCPP)

Cyclic cell-penetrating peptides allows for delivery of otherwiseimpermeable stapled peptides to be efficiently delivered to the cytosoland nucleus of cells. The cCPP of the polypeptide conjugates disclosedherein may be or include any amino sequence which facilitates cellularuptake of the polypeptide conjugates disclosed herein. Suitable cCPPsfor use in the polypeptide conjugates and methods described herein caninclude naturally occurring sequences, modified sequences, and syntheticsequences. In embodiments, the total number of amino acids in the cCPPmay be in the range of from 4 to about 20 amino acids, e.g., about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, and about 19 aminoacids, inclusive of all ranges and subranges therebetween. In someembodiments, the cCPPs disclosed herein comprise about 4 to about toabout 13 amino acids. In particular embodiments, the CPPs disclosedherein comprise about 6 to about 10 amino acids, or about 6 to about 8amino acids.

Each amino acid in the cCPP may independently be a natural ornon-natural amino acid.

In some embodiments, the cCPPs may include any combination of at leasttwo arginines and at least two hydrophobic amino acids. In someembodiments, the cCPPs may include any combination of two to threearginines and at least two hydrophobic amino acids.

In some embodiments, the cCPP used in polypeptide conjugates describedherein has a structure comprising Formula 3:

wherein:

-   -   each of AA₁, AA₂, AA₃, and AA₄, are independently selected from        a D or L amino acid,    -   each of AA_(u) and AA_(z), at each instance and when present,        are independently selected from a D or L amino acid, and    -   m and n are independently selected from a number from 0 to 6;        and

wherein:

-   -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄, and        AA_(z), when present, are independently arginine, and    -   at least two of AA_(u), when present, AA₁, AA₂, AA₃, AA₄, and        AA_(z), when present, are independently a hydrophobic amino        acid.

In some embodiments, each hydrophobic amino acid is independentlyselected from glycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine,homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylicacid, cyclohexylalanine, norleucine, 3-(3-benzothienyl)-alanine,3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine,S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine,3-(1,1′-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyl lysine, eachof which is optionally substituted with one or more substituents. Thestructures of a few of these non-natural aromatic hydrophobic aminoacids (prior to incorporation into the peptides disclosed herein) areprovided below. In particular embodiments, each hydrophobic amino acidis independently a hydrophobic aromatic amino acid. In some embodiments,the aromatic hydrophobic amino acid is naphthylalanine, phenylglycine,homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of whichis optionally substituted with one or more substituents. In particularembodiments, the hydrophobic amino acid is piperidine-2-carboxylic acid,naphthylalanine, tryptophan, or phenylalanine, each of which isoptionally substituted with one or more substituents.

The optional substituent can be any atom or group which does notsignificantly reduce the cytosolic delivery efficiency of the cCPP,e.g., a substituent that does not reduce relative cytosolic deliveryefficiency to less than that of c(FΦRRRRQ). In some embodiments, theoptional substituent can be a hydrophobic substituent or a hydrophilicsubstituent. In certain embodiments, the optional substituent is ahydrophobic substituent. In some embodiments, the substituent increasesthe solvent-accessible surface area (as defined herein) of thehydrophobic amino acid. In some embodiments, the substituent can be ahalogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl,alkylcarbamoyl, alkylcarboxamidyl, alkoxycarbonyl, alkylthio, orarylthio. In some embodiments, the substituent is one or more halogenatoms.

Amino acids having higher hydrophobicity values can be selected toimprove cytosolic delivery efficiency of a cCPP relative to amino acidshaving a lower hydrophobicity value. In some embodiments, eachhydrophobic amino acid independently has a hydrophobicity value which isgreater than that of glycine. In other embodiments, each hydrophobicamino acid independently is a hydrophobic amino acid having ahydrophobicity value which is greater than that of alanine. In stillother embodiments, each hydrophobic amino acid independently has ahydrophobicity value which is greater or equal to phenylalanine.Hydrophobicity may be measured using hydrophobicity scales known in theart. Table 2 below lists hydrophobicity values for various amino acidsas reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A. 1984;81(1):140-144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem.1986; 1986(15):321-53), Kyte and Doolittle (J. Mol. Biol. 1982;157(1):105-132), Hoop and Woods (Proc. Natl. Acad. Sci. U. S. A 1981;78(6):3824-3828), and Janin (Nature. 1979; 277(5696):491-492), theentirety of each of which is herein incorporated by reference in itsentirety. In particular embodiments, hydrophobicity is measured usingthe hydrophobicity scale reported in Engleman, et al.

TABLE 2 Eisenberg Kyrie Hoop Amino and Engleman and and Acid Group Weisset al. Doolittle Woods Janin Ile Nonpolar 0.73 3.1 4.5 −1.8 0.7 PheNonpolar 0.61 3.7 2.8 −2.5 0.5 Val Nonpolar 0.54 2.6 4.2 −1.5 0.6 LeuNonpolar 0.53 2.8 3.8 −1.8 0.5 Trp Nonpolar 0.37 1.9 −0.9 −3.4 0.3 MetNonpolar 0.26 3.4 1.9 −1.3 0.4 Ala Nonpolar 0.25 1.6 1.8 −0.5 0.3 GlyNonpolar 0.16 1.0 −0.4 0.0 0.3 Cys Anti-Polar 0.04 2.0 2.5 −1.0 0.9 TyrAnti-Polar 0.02 −0.7 −1.3 −2.3 −0.4 Pro Nonpolar −0.07 −0.2 −1.6 0.0−0.3 Thr Anti-Polar −0.18 1.2 −0.7 −0.4 −0.2 Ser Anti-Polar −0.26 0.6−0.8 0.3 −0.1 His Charged −0.40 −3.0 −3.2 −0.5 −0.1 Glu Charged −0.62−8.2 −3.5 3.0 −0.7 Asn Anti-Polar −0.64 −4.8 −3.5 0.2 −0.5 GlnAnti-Polar −0.69 −4.1 −3.5 0.2 −0.7 Asp Charged −0.72 −9.2 −3.5 3.0 −0.6Lys Charged −1.10 −8.8 −3.9 3.0 −1.8 Arg Charged −1.80 −12.3 −4.5 3.0−1.4

The chirality of the amino acids can be selected to improve cytosolicuptake efficiency. In some embodiments, at least two of the amino acidshave the opposite chirality. In some embodiments, the at least two aminoacids having the opposite chirality can be adjacent to each other. Insome embodiments, at least three amino acids have alternatingstereochemistry relative to each other. In some embodiments, the atleast three amino acids having the alternating chirality relative toeach other can be adjacent to each other. In some embodiments, at leasttwo of the amino acids have the same chirality. In some embodiments, theat least two amino acids having the same chirality can be adjacent toeach other. In some embodiments, at least two amino acids have the samechirality and at least two amino acids have the opposite chirality. Insome embodiments, the at least two amino acids having the oppositechirality can be adjacent to the at least two amino acids having thesame chirality. Accordingly, in some embodiments, adjacent amino acidsin the cCPP can have any of the following sequences: D-L; L-D; D-L-L-D;L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D.

In some embodiments, an arginine is adjacent to a hydrophobic aminoacid. In some embodiments, the arginine has the same chirality as thehydrophobic amino acid. In some embodiments, at least two arginines areadjacent to each other. In still other embodiments, three arginines areadjacent to each other. In some embodiments, at least two hydrophobicamino acids are adjacent to each other. In other embodiments, at leastthree hydrophobic amino acids are adjacent to each other. In otherembodiments, the cCPPs described herein comprise at least twoconsecutive hydrophobic amino acids and at least two consecutivearginines. In further embodiments, one hydrophobic amino acid isadjacent to one of the arginines. In still other embodiments, the cCPPsdescribed herein comprise at least three consecutive hydrophobic aminoacids and there consecutive arginines. In further embodiments, onehydrophobic amino acid is adjacent to one of the arginines. Thesevarious combinations of amino acids can have any arrangement of D and Lamino acids, e.g., the sequences described above.

In some embodiments, any four adjacent amino acids in the cCPPsdescribed herein (e.g., the cCPPs according to Formula 2) can have oneof the following sequences: AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2), wherein each of AA_(H1) andAA_(H2) are independently a hydrophobic amino acid. Accordingly, in someembodiments, the cCPPs used in the polypeptide conjugates describedherein have a structure according any of Formula 4A-D:

wherein:

each of AA_(H1) and AA_(H2) are independently a hydrophobic amino acid;at each instance and when present, each of AA_(u) and AA_(z) areindependently any amino acid; and

m and n are independently selected from a number from 0 to 6.

In some embodiments, the total number of amino acids (including r, R,AA_(H1), AA_(H2)), in the CPPs of Formula 4-A to 4-D are in the range of6 to 10. In some embodiments, the total number of amino acids is 6. Insome embodiments, the total number of amino acids is 7. In someembodiments, the total number of amino acids is 8. In some embodiments,the total number of amino acids is 9. In some embodiments, the totalnumber of amino acids is 10.

In some embodiments, the sum of m and n is from 2 to 6. In someembodiments, the sum of m and n is 2. In some embodiments, the sum of mand n is 3. In some embodiments, the sum of m and n is 4. In someembodiments, the sum of m and n is 5. In some embodiments, the sum of mand n is 6. In some embodiments, m is 0. In some embodiments, m is 1. Insome embodiments, m is 2. In some embodiments, m is 3. In someembodiments, m is 4. In some embodiments, m is 5. In some embodiments, mis 6. In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2. In some embodiments, n is 3. In some embodiments, nis 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, each hydrophobic amino acid is independentlyselected from glycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, naphthylalanine, phenylglycine,homophenylalanine, tyrosine, cyclohexylalanine, piperidine-2-carboxylicacid, cyclohexylalanine, norleucine, 3-(3-benzothienyl)-alanine,3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine,S-(4-methylbenzyl)cysteine, N-(naphthalen-2-yl)glutamine,3-(1,1′-biphenyl-4-yl)-alanine, tert-leucine, or nicotinoyl lysine, eachof which is optionally substituted with one or more substituents. Inparticular embodiments, each hydrophobic amino acid is independently ahydrophobic aromatic amino acid. In some embodiments, the aromatichydrophobic amino acid is naphthylalanine, phenylglycine,homophenylalanine, phenylalanine, tryptophan, or tyrosine, each of whichis optionally substituted with one or more substituents. In particularembodiments, the hydrophobic amino acid is piperidine-2-carboxylic acid,naphthylalanine, tryptophan, or phenylalanine, each of which isoptionally substituted with one or more substituents.

In some embodiments, each of AA_(H1) and AA_(H2) are independently ahydrophobic amino acid having a hydrophobicity value that is greaterthan that of glycine. In other embodiments, each of AA_(H1) and AA_(H2)are independently a hydrophobic amino acid having a hydrophobicity valuethat is greater than that of alanine. In still other embodiments, eachof AA_(H1) and AA_(H2) are independently a hydrophobic amino acid havinga hydrophobicity value which is greater than that of phenylalanine,e.g., as measured using the hydrophobicity scales described above,including Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A. 1984;81(1):140-144), Engleman, et al. (Ann. Rev. of Biophys. Biophys. Chem.1986; 1986(15):321-53), Kyte and Doolittle (J. Mol. Biol. 1982;157(1):105-132), Hoop and Woods (Proc. Natl. Acad. Sci. U.S.A. 1981;78(6):3824-3828), and Janin (Nature. 1979; 277(5696):491-492), (seeTable 1 above). In particular embodiments, hydrophobicity is measuredusing the hydrophobicity scale reported in Engleman, et al.

The presence of a hydrophobic amino acid on the N- or C-terminal of aD-Arg or L-Arg, or a combination thereof, has also found to improve thecytosolic uptake of the cCPP (and the attached cargo). For example, insome embodiments, the cCPPs disclosed herein may include AA_(H1)-D-Argor D-Arg-AA_(H1). In other embodiments, the cCPPs disclosed herein mayinclude AA_(H1)-L-Arg or L-Arg-AA_(H1).

The size of the hydrophobic amino acid on the N- or C-terminal of theD-Arg or an L-Arg, or a combination thereof (i.e., AA_(H1)), may beselected to improve cytosolic delivery efficiency of the CPP. Forexample, a larger hydrophobic amino acid on the N- or C-terminal of aD-Arg or L-Arg, or a combination thereof, improves cytosolic deliveryefficiency compared to an otherwise identical sequence having a smallerhydrophobic amino acid. The size of the hydrophobic amino acid can bemeasured in terms of molecular weight of the hydrophobic amino acid, thesteric effects of the hydrophobic amino acid, the solvent-accessiblesurface area (SASA) of the side chain, or combinations thereof. In someembodiments, the size of the hydrophobic amino acid is measured in termsof the molecular weight of the hydrophobic amino acid, and the largerhydrophobic amino acid has a side chain with a molecular weight of atleast about 90 g/mol, or at least about 130 g/mol, or at least about 141g/mol. In other embodiments, the size of the amino acid is measured interms of the SASA of the hydrophobic side chain, and the largerhydrophobic amino acid has a side chain with a SASA greater thanalanine, or greater than glycine. In other embodiments, AA_(H1) has ahydrophobic side chain with a SASA greater than or equal to aboutpiperidine-2-carboxylic acid, greater than or equal to about tryptophan,greater than or equal to about phenylalanine, or equal to or greaterthan about naphthylalanine. In some embodiments, AA_(H1) has a sidechain side with a SASA of at least about 200 Å², at least about 210 Å²,at least about 220 Å², at least about 240 Å², at least about 250 Å², atleast about 260 Å², at least about 270 Å², at least about 280 Å², atleast about 290 Å², at least about 300 Å², at least about 310 Å², atleast about 320 Å², or at least about 330 Å². In some embodiments,AA_(H2) has a side chain side with a SASA of at least about 200 Å², atleast about 210 Å², at least about 220 Å², at least about 240 Å², atleast about 250 Å², at least about 260 Å², at least about 270 Å², atleast about 280 Å², at least about 290 Å², at least about 300 Å², atleast about 310 Å², at least about 320 Å², or at least about 330 Å². Insome embodiments, the side chains of AA_(H1) and AA_(H2) have a combinedSASA of at least about 350 Å², at least about 360 Å², at least about 370Å², at least about 380 Å₂, at least about 390 Å², at least about 400 Å²,at least about 410 Å², at least about 420 Å², at least about 430 Å², atleast about 440 Å², at least about 450 Å², at least about 460 Å², atleast about 470 Å², at least about 480 Å², at least about 490 Å²,greater than about 500 Å², at least about 510 Å², at least about 520 Å²,at least about 530 Å², at least about 540 Å², at least about 550 Å², atleast about 560 Å², at least about 570 Å², at least about 580 Å², atleast about 590 Å², at least about 600 Å², at least about 610 Å², atleast about 620 Å², at least about 630 Å², at least about 640 Å²,greater than about 650 Å², at least about 660 Å², at least about 670 Å²,at least about 680 Å², at least about 690 Å², or at least about 700 Å².In some embodiments, AA_(H2) is a hydrophobic amino acid with a sidechain having a SASA that is less than or equal to the SASA of thehydrophobic side chain of AA_(H1). By way of example, and not bylimitation, a cCPP having a Nal-Arg motif exhibits improved cytosolicdelivery efficiency compared to an otherwise identical CPP having aPhe-Arg motif; a cCPP having a Phe-Nal-Arg motif exhibits improvedcytosolic delivery efficiency compared to an otherwise identical cCPPhaving a Nal-Phe-Arg motif, and a phe-Nal-Arg motif exhibits improvedcytosolic delivery efficiency compared to an otherwise identical cCPPhaving a nal-Phe-Arg motif.

As used herein, “hydrophobic surface area” or “SASA” refers to thesurface area (reported as square Ångstroms; Å²) of an amino acid sidechain that is accessible to a solvent. In particular embodiments, SASAis calculated using the ‘rolling ball’ algorithm developed by Shrake &Rupley (J Mol Biol. 79 (2): 351-71), which is herein incorporated byreference in its entirety for all purposes. This algorithm uses a“sphere” of solvent of a particular radius to probe the surface of themolecule. A typical value of the sphere is 1.4 Å, which approximates tothe radius of a water molecule.

SASA values for certain side chains are shown below in Table 3. Incertain embodiments, the SASA values described herein are based on thetheoretical values listed in Table 3 below, as reported by Tien, et al.(PLOS ONE 8(11): e80635. https://doi.org/10.1371/journal.pone.0080635,which is herein incorporated by reference in its entirety for allpurposes.

TABLE 3 SASA values for amino acid side chains. Miller et al. Rose etal. Residue Theoretical Empirical (1987) (1985) Alanine 129.0 121.0113.0 118.1 Arginine 274.0 265.0 241.0 256.0 Asparagine 195.0 187.0158.0 165.5 Aspartate 193.0 187.0 151.0 158.7 Cysteine 167.0 148.0 140.0146.1 Glutamate 223.0 214.0 183.0 186.2 Glutamine 225.0 214.0 189.0193.2 Glycine 104.0 97.0 85.0 88.1 Histidine 224.0 216.0 194.0 202.5Isoleucine 197.0 195.0 182.0 181.0 Leucine 201.0 191.0 180.0 193.1Lysine 236.0 230.0 211.0 225.8 Methionine 224.0 203.0 204.0 203.4Phenylalanine 240.0 228.0 218.0 222.8 Proline 159.0 154.0 143.0 146.8Serine 155.0 143.0 122.0 129.8 Threonine 172.0 163.0 146.0 152.5Tryptophan 285.0 264.0 259.0 266.3 Tyrosine 263.0 255.0 229.0 236.8Valine 174.0 165.0 160.0 164.5

In some embodiments, the cCPP does not include a hydrophobic amino acidon the N- and/or C-terminal of AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2). In alternative embodiments,the cCPP does not include a hydrophobic amino acid having a side chainwhich is larger (as described herein) than at least one of AA_(H1) orAA_(H2). In further embodiments, the cCPP does not include a hydrophobicamino acid with a side chain having a surface area greater than AA_(H1).For example, in embodiments in which at least one of AA_(H1) or AA_(H2)is phenylalanine, the cCPP does not further include a naphthylalanine(although the cCPP include at least one hydrophobic amino acid which issmaller than AA_(H1) and AA_(H2), e.g., leucine). In still otherembodiments, the cCPP does not include a naphthylalanine in addition tothe hydrophobic amino acids in AA_(H2)-AA_(H1)-R-r, AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2), or r-R-AA_(H1)-AA_(H2).

The chirality of the amino acids (i.e., D or L amino acids) can beselected to improve cytosolic delivery efficiency of the cCPP (and theattached cargo as described below). In some embodiments, the hydrophobicamino acid on the N- or C-terminal of an arginine (e.g., AA_(H1)) hasthe same or opposite chirality as the adjacent arginine. In someembodiments, AA_(H1) has the opposite chirality as the adjacentarginine. For example, when the arginine is D-arg (i.e. “r”), AA_(H1) isa D-AA_(H1), and when the arginine is L-Arg (i.e., “R”), AA_(H1) is aL-AA_(H1). Accordingly, in some embodiments, the cCPPs disclosed hereinmay include at least one of the following motifs: D-AA_(H1)-D-arg,D-arg-D-AA_(H1), L-AA_(H1)-L-Arg, or L-Arg-LAA_(H1). In particularembodiments, when arginine is D-arg, AA_(H) can be D-nal, D-trp, orD-phe. In another non-limiting example, when arginine is L-Arg, AA_(H)can be L-Nal, L-Trp, or L-Phe.

In some embodiments, the cCPPs described herein include three arginines.Accordingly, in some embodiments, the cCPPs described herein include oneof the following sequences: AA_(H2)-AA_(H1)-R-r-R,AA_(H2)-AA_(H1)-R-r-r, AA_(H2)-AA_(H1)-r-R-R, AA_(H2)-AA_(H1)-r-R-r,R-R-r-AA_(H1)-AA_(H2), r-R-r-AA_(H1)-AA_(H2), r-r-R-AA_(H1)-AA_(H2), or,R-r-R-AA_(H1)-AA_(H2). In particular embodiments, the cCPPs have one ofthe following sequences AA_(H2)-AA_(H1)-R-r-R, AA_(H2)-AA_(H1)-r-R-r,r-R-r-AA_(H1)-AA_(H2), or R-r-R-AA_(H1)-AA_(H2). In some embodiments,the chirality of AA_(H1) and AA_(H2) can be selected to improvecytosolic uptake efficiency, e.g., as described above, where AA_(H1) hasthe same chirality as the adjacent arginine, and AA_(H1) and AA_(H2)have the opposite chirality.

In some embodiments, the cCPPs described herein include threehydrophobic amino acids. Accordingly, in some embodiments, the cCPPsdescribed herein include one of the following sequences:AA_(H3)-AA_(H2)-AA_(H1)-R-r, AA_(H3)-AA_(H2)-AA_(H1)-R-r,AA_(H3)-AA_(H2)-AA_(H1)-r-R, AA_(H3)-AA_(H2)-AA_(H1)-r-R,R-r-AA_(H1)-AA_(H2)-AA_(H3), R-r-AA_(H1)-AA_(H2)-AA_(H3),r-R-AA_(H1)-AA_(H2)-AA_(H3), or, r-R-AA_(H1)-AA_(H2)-AA_(H3), whereinAA_(H3) is any hydrophobic amino acid described above, e.g.,piperidine-2-carboxylic acid, naphthylalanine, tryptophan, orphenylalanine. In some embodiments, the chirality of AA_(H1), AA_(H2),and AA_(H3) can be selected to improve cytosolic uptake efficiency,e.g., as described above, where AA_(H1) has the same chirality as theadjacent arginine, and AA_(H1) and AA_(H2) have the opposite chirality.In other embodiments, the size of AA_(H1), AA_(H2), and AA_(H3) can beselected to improve cytosolic uptake efficiency, e.g., as describedabove, where AA_(H3) has a SAS of less than or equal to AA_(H1) and/orAA_(H2).

In some embodiments, AA_(H1) and AA_(H2) have the same or oppositechirality. In certain embodiments, AA_(H1) and AA_(H2) have the oppositechirality. Accordingly, in some embodiments, the cCPPs disclosed hereininclude at least one of the following sequences:D-AA_(H2)-L-AA_(H1)-R-r; L-AA_(H2)-D-AA_(H1)-r-R;R-r-D-AA_(H1)-L-AA_(H2); or r-R-L-AA_(H1)-D-AA_(H1), wherein each ofD-AA_(H1) and D-AA_(H2) is a hydrophobic amino acid having a Dconfiguration, and each of L-AA_(H1) and L-AA_(H2) is a hydrophobicamino acid having an L configuration. In some embodiments, each ofD-AA_(H1) and D-AA_(H2) is independently selected from the groupconsisting of D-pip, D-nal, D-trp, and D-phe. In particular embodiments,D-AA_(H1) or D-AA_(H2) is D-nal. In other particular embodiments,D-AA_(H1) is D-nal. In some embodiments, each of L-AA_(H1) and L-AA_(H2)is independently selected from the group consisting of L-Pip, L-Nal,L-Trp, and L-Phe. In particular embodiments, each of L-AA_(H1) andL-AA_(H2) is L-Nal.

As discussed above, the disclosure provides for various modifications toa cyclic peptide sequence, which may improve cytosolic deliveryefficiency. In some embodiments, improved cytosolic uptake efficiencycan be measured by comparing the cytosolic delivery efficiency of theCPP having the modified sequence to a proper control sequence. In someembodiments, the control sequence does not include a particularmodification (e.g., matching chirality of R and AA_(H1)) but isotherwise identical to the modified sequence. In other embodiments, thecontrol has the following sequence: cyclic(FΦRRRRQ)

As used herein cytosolic delivery efficiency refers to the ability of acCPP to traverse a cell membrane and enter the cytosol. In embodiments,cytosolic delivery efficiency of the cCPP is not dependent on a receptoror a cell type. Cytosolic delivery efficiency can refer to absolutecytosolic delivery efficiency or relative cytosolic delivery efficiency.

Absolute cytosolic delivery efficiency is the ratio of cytosolicconcentration of a cCPP (or a polypeptide conjugate) over theconcentration of the cCPP (or the polypeptide conjugate) in the growthmedium. Relative cytosolic delivery efficiency refers to theconcentration of a cCPP in the cytosol compared to the concentration ofa control cCPP in the cytosol. Quantification can be achieved byfluorescently labeling the cCPP (e.g., with a FITC dye) and measuringthe fluorescence intensity using techniques well-known in the art.

In particular embodiments, relative cytosolic delivery efficiency isdetermined by comparing (i) the amount of a cCPP of the inventioninternalized by a cell type (e.g., HeLa cells) to (ii) the amount of thecontrol cCPP internalized by the same cell type. To measure relativecytosolic delivery efficiency, the cell type may be incubated in thepresence of a cell-penetrating peptide of the invention for a specifiedperiod of time (e.g., 30 minutes, 1 hour, 2 hours, etc.) after which theamount of the cCPP internalized by the cell is quantified using methodsknown in the art, e.g., fluorescence microscopy. Separately, the sameconcentration of the control cCPP is incubated in the presence of thecell type over the same period of time, and the amount of the controlcCPP internalized by the cell is quantified.

In other embodiments, relative cytosolic delivery efficiency can bedetermined by measuring the IC₅₀ of a cCPP having a modified sequencefor an intracellular target, and comparing the IC₅₀ of the cCPP havingthe modified sequence to a proper control sequence (as describedherein).

In some embodiments, the relative cytosolic delivery efficiency of thecCPPs described herein in the range of from about 1% to about 1000%compared to, e.g., cyclo(FΦRRRRQ) or a linear cell-penetrating peptidesequence (such as HIV-TAT, a polyarginine sequence, and the like), e.g.,about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%,about 120%, about 130%, about 140%, about 150%, about 160%, about 170%,about 180%, about 190%, about 200%, about 210%, about 220%, about 230%,about 240%, about 250%, about 260%, about 270%, about 280%, about 290%,about 300%, about 310%, about 320%, about 330%, about 340%, about 350%,about 360%, about 370%, about 380%, about 390%, about 400%, about 410%,about 420%, about 430%, about 440%, about 450%, about 460%, about 470%,about 480%, about 490%, about 500%, about 510%, about 520%, about 530%,about 540%, about 550%, about 560%, about 570%, about 580%, or about590%, about 600%, about 610%, about 620%, about 630%, about 640%, about650%, about 660%, about 670%, about 680%, about 690%, about 700%, about710%, about 720%, about 730%, about 740%, about 750%, about 760%, about770%, about 780%, or about 790%, about 800%, about 810%, about 820%,about 830%, about 840%, about 850%, about 860%, about 870%, about 880%,about 890%, about 900%, about 910%, about 920%, about 930%, about 940%,about 950%, about 960%, about 970%, about 980%, or about 1000%,inclusive of all values and subranges therebetween.

In other embodiments, the absolute cytosolic delivery efficacy of fromabout 40% to about 100%, e.g., about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, inclusive of all values and subrangestherebetween.

Non-limiting examples of suitable cyclic cell penetrating peptides areprovided in Table 4.

TABLE 4 Examples of cyclic cell penetrating peptides. ID cCPP SequencePCT 1 cyclo(FΦRRRQ) PCT 2 cyclo(FΦRRRC) PCT 3 cyclo(FΦRRRU) PCT 4cyclo(RRRΦFQ) PCT 5 cyclo(RRRRΦF) PCT 6 cyclo(FΦRRRR) PCT 7cyclo(FΦrRrRq) PCT 8 cyclo(FΦrRrRQ) PCT 9 cyclo(FΦRRRRQ) PCT 10cyclo(fΦRrRrQ) PCT 11 cyclo(RRFRΦRQ) PCT 12 cyclo(FRRRRΦQ) PCT 13cyclo(rRFRΦRQ) PCT 14 cyclo(RRΦFRRQ) PCT 15 cyclo(CRRRRFWQ) (SEQ ID NO:2, underlined portion only) PCT 16 cyclo(FfΦRrRrQ) PCT 17cyclo(FFΦRRRRQ) PCT 18 cyclo(RFRFRΦRQ) PCT 19 cyclo(URRRRFWQ) (SEQ IDNO: 3, underlined portion only) PCT 20 cyclo(CRRRRFWQ) (SEQ ID NO: 4,underlined portion only) PCT 21 cyclo(FΦRRRRQK) PCT 22 cyclo(FΦRRRRQC)PCT 23 cyclo(fΦRrRrRQ) PCT 24 cyclo(FΦRRRRRQ) PCT 25 cyclo(RRRRΦFDΩC)PCT 26 cyclo(FΦRRR) PCT 27 cyclo(FWRRR) (SEQ ID NO: 5, underlinedportion only) PCT 28 cyclo(RRRΦF) PCT 29 cyclo(RRRWF) (SEQ ID NO: 6,underlined portion only) SAR 1 cyclo(FΦRRRRQ) SAR 19 cyclo(FFRRRQ) (SEQID NO: 7, underlined portion only) SAR 20 cyclo(FFrRrQ) SAR 21cyclo(FFRrRQ) SAR 22 cyclo(FRFRRQ) (SEQ ID NO: 8, underlined portiononly) SAR 23 cyclo(FRRFRQ) (SEQ ID NO: 9, underlined portion only) SAR24 cyclo(FRRRFQ) (SEQ ID NO: 10, underlined portion only) SAR 25cyclo(GΦRRRQ) SAR 26 cyclo(FFFRAQ) (SEQ ID NO: 11, underlined portiononly) SAR 27 cyclo(FFFRRQ) (SEQ ID NO: 12, underlined portion only) SAR28 cyclo(FFRRRRQ) (SEQ ID NO: 13, underlined portion only) SAR 29cyclo(FRRFRRQ) (SEQ ID NO: 14, underlined portion only) SAR 30cyclo(FRRRFRQ) (SEQ ID NO: 15, underlined portion only) SAR 31cyclo(RFFRRRQ) (SEQ ID NO: 16, underlined portion only) SAR 32cyclo(RFRRFRQ) (SEQ ID NO: 17, underlined portion only) SAR 33cyclo(FRFRRRQ) (SEQ ID NO: 18, underlined portion only) SAR 34cyclo(FFFRRRQ) (SEQ ID NO: 19, underlined portion only) SAR 35cyclo(FFRRRFQ) (SEQ ID NO: 20, underlined portion only) SAR 36cyclo(FRFFRRQ) (SEQ ID NO: 21, underlined portion only) SAR 37cyclo(RRFFFRQ) (SEQ ID NO: 22, underlined portion only) SAR 38cyclo(FFRFRRQ) (SEQ ID NO: 23, underlined portion only) SAR 39cyclo(FFRRFRQ) (SEQ ID NO: 24, underlined portion only) SAR 40cyclo(FRRFFRQ) (SEQ ID NO: 25, underlined portion only) SAR 41cyclo(FRRFRFQ) (SEQ ID NO: 26, underlined portion only) SAR 42cyclo(FRFRFRQ) (SEQ ID NO: 27, underlined portion only) SAR 43cyclo(RFFRFRQ) (SEQ ID NO: 28, underlined portion only) SAR 44cyclo(GΦRRRRQ) SAR 45 cyclo(FFFRRRRQ) (SEQ ID NO: 29, underlined portiononly) SAR 46 cyclo(RFFRRRRQ) (SEQ ID NO: 30, underlined portion only)SAR 47 cyclo(RRFFRRRQ) (SEQ ID NO: 31, underlined portion only) SAR 48cyclo(RFFFRRRQ) (SEQ ID NO: 32, underlined portion only) SAR 49cyclo(RRFFFRRQ) (SEQ ID NO: 33, underlined portion only) SAR 50cyclo(FFRRFRRQ) (SEQ ID NO: 34, underlined portion only) SAR 51cyclo(FFRRRRFQ) (SEQ ID NO: 35, underlined portion only) SAR 52cyclo(FRRFFRRQ) (SEQ ID NO: 36, underlined portion only) SAR 53cyclo(FFFRRRRRQ) (SEQ ID NO: 37, underlined portion only) SAR 54cyclo(FFFRRRRRRQ) (SEQ ID NO: 38, underlined portion only) SAR 55cyclo(FΦRrRrQ) SAR 56 cyclo(XXRRRRQ) (SEQ ID NO: 39, underlined portiononly) SAR 57 cyclo(FfFRrRQ) SAR 58 cyclo(fFfrRrQ) SAR 59 cyclo(fFfRrRQ)SAR 60 cyclo(FfFrRrQ) SAR 61 cyclo(fFΦrRrQ) SAR 62 cyclo(fΦfrRrQ) SAR 63cyclo(ΦFfrRrQ) SAR 64 cyclo(FΦrRrQ) SAR 65 cyclo(fΦrRrQ) SAR 66Ac-(Lys-fFRrRrD) SAR 67 Ac-(Dap-fFRrRrD) SAR 68

SAR 69

SAR 70

SAR 71

Pin1 15 cyclo(Pip-Nal-Arg-Glu-arg-arg-glu) Pin1 16cyclo(Pip-Nal-Arg-Arg-arg-arg-glu) Pin1 17cyclo(Pip-Nal-Nal-Arg-arg-arg-glu) Pin1 18cyclo(Pip-Nal-Nal-Arg-arg-arg-Glu) Pin1 19cyclo(Pip-Nal-Phe-Arg-arg-arg-glu) Pin1 20cyclo(Pip-Nal-Phe-Arg-arg-arg-Glu) Pin1 21cyclo(Pip-Nal-phe-Arg-arg-arg-glu) Pin1 22cyclo(Pip-Nal-phe-Arg-arg-arg-Glu) Pin1 23cyclo(Pip-Nal-nal-Arg-arg-arg-Glu) Pin1 24cyclo(Pip-Nal-nal-Arg-arg-arg-glu) Rev-13 [Pim-RQRR-Nlys]GRRR^(b) hLF

cTat [KrRrGrKkRrE]^(c) cR10 [KrRrRrRrRrRE]^(c) L-50 [RVRTRGKRRIRRpP]L-51 [RTRTRGKRRIRVpP] [WR]₄ [WRWRWRWR] MCoTI-II

Rotstein [P-Cha-r-Cha-r-Cha-r-Cha-r-G]^(d) et al. Chem. Eur. J. 2011Lian et Tm(SvP-F₂Pmp-H)-Dap-(FΦRRRR-Dap)]^(f) al. J. Am. Chem. Soc. 2014Lian et [Tm(a-Sar-D-pThr-Pip-ΦRAa)-Dap-(FΦRRR-Dap)]^(f) al. J. Am. Chem.Soc. 2014 IA8b [CRRSRRGCGRRSRRCG]^(g) Dod- [K(Dod)RRRR] [R₅] LK-3

RRRR-[KRRRE]^(c) RRR-[KRRRRE]^(c) RR-[KRRRRRE]^(c) R-[KRRRRRRE]^(c)[CR]₄ [CRCRCRCR] cyc3 [Pra-LRKRLRKFRN-AzK]^(h) PMBT-Dap-[Dap-Dap-f-L-Dap-Dap-T] GPMB T-Agp-[Dap-Agp-f-L-Agp-Agp-T] cCPP1cyclo(FΦRRRRQ) cCPP12 cyclo(FfΦRrRrQ) cCPP9 cyclo(fΦRrRrQ) cCPP11cyclo(fΦRrRrRQ) cCPP18 cyclo(FΦrRrRq) cCPP13 cyclo(FΦrRrRQ) cCPP6cyclo(FΦRRRRRQ) cCPP3 cyclo(RRFRΦRQ) cCPP7 cyclo(FFΦRRRRQ) cCPP8cyclo(RFRFRΦRQ) cCPP5 cyclo(FΦRRRQ) cCPP4 cyclo(FRRRRΦQ) cCPP10cyclo(rRFRΦRQ) cCPP2 cyclo(RRΦFRRQ) Φ, L-2-naphthylalanine; Pim, pimelicacid; Nlys, lysine peptoid residue; D-pThr, D-phosphothreonine; Pip,L-piperidine-2-carboxylic acid; Cha, L-3-cyclohexyl-alanine; Tm,trimesic acid; Dap, L-2,3-diaminopropionic acid; Sar, sarcosine; F₂Pmp,L-difluorophosphonomethyl phenylalanine; Dod, dodecanoyl; Pra,L-propargylglycine; AzK, L-6-Azido-2-amino-hexanoic; Agp,L-2-amino-3-guanidinylpropionic acid; ^(b)Cyclization between Pim andNlys; ^(c)Cyclization between Lys and Glu; ^(d)Macrocyclization bymulticomponent reaction with aziridine aldehyde and isocyanide;^(e)Cyclization between the main-chain of Gln residue; ^(f)N-terminalamine and side chains of two Dap residues bicyclized with Tm; ^(g)ThreeCys side chains bicyclized with tris(bromomethyl)benzene;^(h)Cyclization by the click reaction between Pra and Azk.

Additionally, the cCPP used in the polypeptide conjugates and methodsdescribed herein can include any sequence disclosed in: U.S. applicationSer. No. 15/312,878 (US Pub. No. US 2017/0190743 A1); U.S. applicationSer. No. 15/360,719 (US Pub. No. US 2017/0355730); PCT/US2017/060881(and the resulting US publication); and PCT/US2017/062951 (and theresulting US publication), each of which is incorporated by reference inits entirety for all purposes.

In some embodiments, the cCPP improves the cytosolic delivery efficiencyby about 1.1 fold to about 30 fold, compared to a linearcell-penetrating peptide sequence (such as HIV-TAT, polyarginine and thelike), e.g., about 1.2, about 1.3, about 1.4, about 1.5, about 1.6,about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5,about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about13.5, about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about19.5, about 20, about 20.5, about 21.0, about 21.5, about 22.0, about22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0, about25.5, about 26.0, about 26.5, about 27.0, about 27.5, about 28.0, about28.5, about 29.0, or about 29.5 fold, inclusive of all values andsubranges therebetween.

Linker

As discussed above, the cCPP may be directly conjugated to the stapledpeptide (e.g., by a covalent bond between a side chain of an amino acidon the cCPP and an appropriate group on the stapled peptide) or a linkermay be used to conjugate the cCPP to the stapled peptide. As usedherein, “linker” refers to a moiety that forms a covalent bond betweenthe two or more components of the polypeptide conjugates disclosedherein (e.g., a cCPP and a stapled peptide via the staple or thepeptide).

In various embodiments, the linker is covalently bound to an amino acidon the cCPP and either an amino acid on the peptide or the staple. Thelinker may be any moiety which conjugates two or more of the cCPPmoiety, the peptide, and the staple. In some embodiments, the linker canbe an amino acid. In other embodiments, the precursor to the linker canbe any appropriate molecule which is capable of forming two or morebonds with amino acids in the cCPP, the peptide, the staple, andcombinations thereof. Thus, in various embodiments, the precursor of thelinker has two or more functional groups, each of which are capable offorming a covalent bond to at least two of the cCPP moiety, the peptide,and the staple. For example, the linker can be covalently bound to theN-terminus, C-terminus, or side chain, or combinations thereof, of anyamino acid in the cCPP moiety, the peptide, or the staple. In particularembodiments, the linker forms a covalent bond between the cCPP andpeptide.

In some embodiments, the linker is selected from the group consisting ofat least one amino acid, alkylene, alkenylene, alkynylene, aryl,cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, heteroaryl, ether,each of which can be optionally substituted as defined above.Non-limiting examples of linkers include polyethylene glycol, optionallyconjugated to a lysine residue.

In some embodiments, the linker is covalently bound to the N orC-terminus of an amino acid on the stapled peptide, or to a side chainof glutamine, asparagine, or lysine, or a modified side chain ofglutamine or asparagine (e.g., a reduced side chain having an aminogroup), on the cCPP, peptide, or staple. In particular embodiments, thelinker forms a bond with the side chain of glutamine on the cCPP. Inother particular embodiments, the linker described herein has astructure of L-1 or L-2:

wherein

-   -   AA_(s) is a side chain or terminus of an amino acid on the        peptide or staple;    -   AA_(c) is a side chain or terminus of an amino acid of the cCPP;    -   p is an integer from 0 to 10; and    -   q is an integer from 1 to 50.

In some embodiments, the linker is capable of releasing the stapledpeptide from the cCPP after the polypeptide conjugate enters the cytosolof the cell. In some embodiments, the linker contains a group, or formsa group after binding to cCPP, peptide, staple, or a combinationthereof, that is cleaved after cytosolic uptake of the polypeptideconjugate to thereby release the peptide. Non-limiting examples ofphysiologically cleavable linking group include carbonate,thiocarbonate, thioester, disulfide, sulfoxide, hydrazine,protease-cleavable dipeptide linker, and the like.

For example, in embodiments, the linker is covalently bound to thestapled peptide through a disulfide bond e.g., with the side chain ofcysteine or cysteine analog located in the stapled peptide or the cCPP.In some embodiments, the disulfide bond is formed between a thiol groupon a precursor of the linker, and the side chain of cysteine or an aminoacid analog having a thiol group on the peptide, wherein the bond tohydrogen on each of the thiol groups is replaced by a bond to a sulfuratom. Non-limiting examples of amino acid analogs having a thiol groupwhich can be used with the polypeptide conjugates disclosed herein arediscussed above.

Methods of Treatment

As discussed above, the polypeptide conjugates described herein can beused to treat or prevent a disease, disorder, or condition in a patientin need thereof. In some embodiments, treatment refers to partial orcomplete alleviation, amelioration, relief, inhibition, delaying onset,reducing severity and/or incidence of the disease, disorder, orcondition in the patient.

The terms, “improve,” “increase,” “reduce,” “decrease,” and the like, asused herein, indicate values that are relative to a control. In someembodiments, a suitable control is a baseline measurement, such as ameasurement in the same individual prior to initiation of the treatmentdescribed herein, or a measurement in a control individual (or multiplecontrol individuals) in the absence of the treatment described herein.

The individual (also referred to as “patient”) being treated is anindividual (fetus, infant, child, adolescent, or adult human) having adisease, disorder, or condition, or having the potential to develop adisease, disorder, or condition.

In some embodiments, the individual is an individual who has beenrecently diagnosed with a disease, disorder or condition. Typically,early treatment (treatment commencing as soon as possible afterdiagnosis) is important to minimize the effects of the disease, disorderor condition and to maximize the benefits of treatment.

In some embodiments, the polypeptide conjugates may be used to treat anindividual diagnosed with a cancer. The polypeptide conjugates of theinstant invention may be used to treat, for example, the followingcancers: brain tumors such as for example acoustic neurinoma,astrocytomas such as fibrillary, protoplasmic, gemistocytic, anaplastic,pilocytic astrocytomas, glioblastoma, gliosarcoma, pleomorphicxanthoastrocytoma, subependymal large-cell giant cell astrocytoma anddesmoplastic infantile astrocytoma; brain lymphomas, brain metastases,hypophyseal tumor such as prolactinoma, hypophyseal incidentaloma, HGH(human growth hormone) producing adenoma and corticotropic adenoma,craniopharyngiomas, medulloblastoma, meningioma and oligodendroglioma;nerve tumors such as for example tumors of the vegetative nervous systemsuch as neuroblastoma, ganglioneuroma, paraganglioma (pheochromocytoma,chromaffinoma) and glomus-caroticum tumor, tumors on the peripheralnervous system such as amputation neuroma, neurofibroma, neurinoma(neurilemmoma, Schwannoma) and malignant Schwannoma, as well as tumorsof the central nervous system such as brain and bone marrow tumors;intestinal cancer such as for example carcinoma of the rectum, colon,anus and duodenum; eyelid tumors (basalioma or adenocarcinoma of theeyelid apparatus); retinoblastoma; carcinoma of the pancreas; carcinomaof the bladder; lung tumors (bronchial carcinoma—small-cell lung cancer(SCLC), non-small-cell lung cancer (NSCLC) such as for examplespindle-cell plate epithelial carcinomas, adenocarcinomas (acinary,papillary, bronchiolo-alveolar) and large-cell bronchial carcinoma(giant cell carcinoma, clear-cell carcinoma)); breast cancer such asductal, lobular, mucinous or tubular carcinoma, Paget's carcinoma;non-Hodgkin's lymphomas (B-lymphatic or T-lymphatic NHL) such as forexample hair cell leukemia, Burkitt's lymphoma or mycosis fungoides;Hodgkin's disease; uterine cancer (corpus carcinoma or endometrialcarcinoma); CUP syndrome (Cancer of Unknown Primary); ovarian cancer(ovarian carcinoma-mucinous or serous cystoma, endometriodal tumors,clear cell tumor, Brenner's tumor); gall bladder cancer; bile ductcancer such as for example Klatskin tumor; testicular cancer (germinalor non-germinal germ cell tumors); laryngeal cancer such as for examplesupra-glottal, glottal and subglottal tumors of the vocal cords; bonecancer such as for example osteochondroma, chondroma, chondroblastoma,chondromyxoid fibroma, chondrosarcoma, osteoma, osteoid osteoma,osteoblastoma, osteosarcoma, non-ossifying bone fibroma, osteofibroma,desmoplastic bone fibroma, bone fibrosarcoma, malignant fibroushistiocytoma, osteoclastoma or giant cell tumor, Ewing's sarcoma, andplasmocytoma, head and neck tumors (HNO tumors) such as for exampletumors of the lips, and oral cavity (carcinoma of the lips, tongue, oralcavity), nasopharyngeal carcinoma (tumors of the nose,lymphoepithelioma), pharyngeal carcinoma, oropharyngeal carcinomas,carcinomas of the tonsils (tonsil malignant) and (base of the) tongue,hypopharyngeal carcinoma, laryngeal carcinoma (cancer of the larynx),tumors of the paranasal sinuses and nasal cavity, tumors of the salivaryglands and ears; liver cell carcinoma (hepatocellular carcinoma (HCC);leukemias, such as for example acute leukemias such as acutelymphatic/lymphoblastic leukemia (ALL), acute myeloid leukemia (AML);chronic lymphatic leukemia (CLL), chronic myeloid leukemia (CML);stomach cancer (papillary, tubular or mucinous adenocarcinoma,adenosquamous, squamous or undifferentiated carcinoma; malignantmelanomas such as for example superficially spreading (SSM), nodular(NMM), lentigo-maligna (LMM), acral-lentiginous (ALM) or amelanoticmelanoma (AMM); renal cancer such as for example kidney cell carcinoma(hypernephroma or Grawitz's tumor); oesophageal cancer; penile cancer;prostate cancer; vaginal cancer or vaginal carcinoma; thyroid carcinomassuch as for example papillary, follicular, medullary or anaplasticthyroid carcinoma; thymus carcinoma (thymoma); cancer of the urethra(carcinoma of the urethra, urothelial carcinoma) and cancer of thevulva.

In other embodiments, the polypeptide conjugates may be used to treat aninflammatory disease or disorder. The inflammatory disease or disordermay be a respiratory disease such as, for example, asthma or chronicobstructive pulmonary disease, a chronic degenerative disease such asrheumatoid arthritis, osteoarthritis or osteoporosis, a dermatologicalcondition such as psoriasis, scleroderma, atopic dermatitis, ichthyosis,pemphigus, acne, skin aging or wrinkles, a chronic demyelinating diseasesuch as multiple sclerosis; an inflammatory bowel disease such asulcerative colitis or Crohn's disease; a dental disease such asperiodontal disease or gingivitis; an inflammatory nail disease such asnail psoriasis; lichen planus, alopecia areata, systemic lupuserythematosus, diabetic nephropathy, lupus nephritis, IgA nephropathy orglomerulonephritis, graft versus host disease or an ophthalmiccondition.

In other embodiments, the polypeptide conjugates are used to treat anautoimmune disease or condition. The autoimmune disease or condition maybe insulin-dependent diabetes mellitus, multiple sclerosis, rheumatoidarthritis, autoimmune uveitis, primary biliary cirrhosis, myastheniagravis, Sjogren's syndrome, pemphigus vulgaris, scleroderma, perniciousanemia, systemic lupus erythematosus, Grave's disease, inflammatorybowel disease, celiac disease, autoimmune thyroid disease such asHashimoto's disease, autoimmune liver disease, Addison's disease,transplant rejection, graft vs. host disease, host vs. graft disease,ankylosing spondylitis, Chagas disease, chronic obstructive pulmonarydisease, Crohns Disease, dermatomyositis, endometriosis, Goodpasture'ssyndrome, Guillain-Barre syndrome (GBS), hidradenitis suppurativa,Kawasaki disease, IgA nephropathy, idiopathic thrombocytopenic purpura,interstitial cystitis, mixed connective tissue disease, morphea,narcolepsy, neuromyotonia, psoriasis, psoriatic arthritis, polymyositis,relapsing polychondritis, sarcoidosis, schizophrenia, stiff personsyndrome, temporal arteritis, ulcerative colitis, vasculitis, vitiligo,Wegener's granulomatosis, and combinations thereof.

The polypeptide conjugates provided herein can treat the above-describeddiseases, disorders, or conditions, for instance, by disrupting nativeprotein-protein, protein-ligand, and/or protein-receptor interactions.For example, many biologically important protein/protein interactions,such as p53/MDM2 and Bcl-X1/Bak, are mediated by one protein donating ahelix into a cleft of its helix-accepting partner. The interaction ofp53 and MDM2 and mutations in the p53 gene have been identified invirtually half of all reported cancer cases (see, Shair Chem. & Biol.1997, 4, 791, the entire contents of which are incorporated herein byreference). As stresses are imposed on a cell, p53 is believed toorchestrate a response that leads to either cell-cycle arrest and DNArepair, or programmed cell death. As well as mutations in the p53 genethat alter the function of the p53 protein directly, p53 can be alteredby changes in MDM2. The MDM2 protein has been shown to bind to p53 anddisrupt transcriptional activation by associating with thetransactivation domain of p53. For example, an 11 amino-acid peptidederived from the transactivation domain of p53 forms an amphipathicalpha-helix of 2.5 turns that inserts into the MDM2 crevice.

Combination Therapies

In some embodiments, the polypeptide conjugates disclosed herein can beadministered in combination with other therapies. The polypeptideconjugates can be administered simultaneous, sequentially, or atdistinct time points as part of the same therapeutic regimen.

In some embodiments, the polypeptide conjugates disclosed herein areadministered in combination with one or more chemotherapeutic agents.Chemotherapeutic agents which may be administered in combination withthe compounds according to the invention include, without beingrestricted thereto, hormones, hormone analogues and antihormones (e.g.tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate,flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproteroneacetate, finasteride, buserelin acetate, fludrocortisone,fluoxymesterone, medroxyprogesterone, octreotide), aromatase inhibitors(e.g. anastrozole, letrozole, liarozole, vorozole, exemestane,atamestane), LHRH agonists and antagonists (e.g. goserelin acetate,leuprolide), inhibitors of growth factors (growth factors such as forexample “platelet derived growth factor” and “hepatocyte growth factor”,inhibitors are for example “growth factor” antibodies, “growth factorreceptor” antibodies and tyrosinekinase inhibitors, such as for examplegefitinib, lapatinib and trastuzumab); signal transduction inhibitors(e.g. imatinib and sorafenib); antimetabolites (e.g. antifolates such asmethotrexate, pemetrexed and raltitrexed, pyrimidine analogues such as5-fluorouracil, capecitabin and gemcitabin, purine and adenosineanalogues such as mercaptopurine, thioguanine, cladribine andpentostatin, cytarabine, fludarabine); antitumour antibiotics (e.g.anthracyclins such as doxorubicin, daunorubicin, epirubicin andidarubicin, mitomycin-C, bleomycin, dactinomycin, plicamycin,streptozocin); platinum derivatives (e.g. cisplatin, oxaliplatin,carboplatin); alkylation agents (e.g. estramustin, mechlorethamine,melphalan, chlorambucil, busulphan, dacarbazin, cyclophosphamide,ifosfamide, temozolomide, nitrosoureas such as for example carmustin andlomustin, thiotepa); antimitotic agents (e.g. Vinca alkaloids such asfor example vinblastine, vindesin, vinorelbin and vincristine; andtaxanes such as paclitaxel, docetaxel); topoisomerase inhibitors (e.g.epipodophyllotoxins such as for example etoposide and etopophos,teniposide, amsacrin, topotecan, irinotecan, mitoxantron) and variouschemotherapeutic agents such as amifostin, anagrelid, clodronat,filgrastim, interferon alpha, leucovorin, rituximab, procarbazine,levamisole, mesna, mitotane, pamidronate and porfimer.

Methods of Making

The polypeptide conjugates described herein can be prepared in a varietyof ways known to one skilled in the art of organic synthesis orvariations thereon as appreciated by those skilled in the art. Thecompounds described herein can be prepared from readily availablestarting materials. Optimum reaction conditions can vary with theparticular reactants or solvents used, but such conditions can bedetermined by one skilled in the art.

Variations on the compounds described herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high-performance liquidchromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesiswherein the amino acid α-N-terminal is protected by an acid or baseprotecting group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the disclosed compounds. Other preferredside chain protecting groups are, for side chain amino groups likelysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Solid supports forsynthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° C. and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.One method for coupling to the deprotected 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer. In one example, the α-N-terminal inthe amino acids of the growing peptide chain are protected with Fmoc.The removal of the Fmoc protecting group from the α-N-terminal side ofthe growing peptide is accomplished by treatment with a secondary amine,preferably piperidine. Each protected amino acid is then introduced inabout 3-fold molar excess, and the coupling is preferably carried out inDMF. The coupling agent can beO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamine.Alternatively, the peptide can be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide can be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups can beaccomplished using the cleavage cocktail described above. The fullydeprotected peptide can be purified by a sequence of chromatographicsteps employing any or all of the following types: ion exchange on aweakly basic resin (acetate form); hydrophobic adsorption chromatographyon underderivatized polystyrene-divinylbenzene (for example, AmberliteXAD); silica gel adsorption chromatography; ion exchange chromatographyon carboxymethylcellulose; partition chromatography, e.g. on SephadexG-25, LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

Methods of Administration

In vivo application of the disclosed polypeptide conjugates, andcompositions containing them, can be accomplished by any suitable methodand technique presently or prospectively known to those skilled in theart. For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral andparenteral routes of administration. As used herein, the term parenteralincludes subcutaneous, intradermal, intravenous, intramuscular,intraperitoneal, and intrasternal administration, such as by injection.Administration of the disclosed compounds or compositions can be asingle administration, or at continuous or distinct intervals as can bereadily determined by a person skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 100% by weight of the total of one ormore of the subject compounds based on the weight of the totalcomposition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 2003/0032594 and 2002/0120100 discloseamino acid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publication No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy) propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL); chondroitin;chitin; and chitosan.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts or prodrugs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptoms ordisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counterindications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. Pharmaceutical compositions adapted for oral, topical orparenteral administration, comprising an amount of a compound constitutea preferred aspect. The dose administered to a patient, particularly ahuman, should be sufficient to achieve a therapeutic response in thepatient over a reasonable time frame, without lethal toxicity, andpreferably causing no more than an acceptable level of side effects ormorbidity. One skilled in the art will recognize that dosage will dependupon a variety of factors including the condition (health) of thesubject, the body weight of the subject, kind of concurrent treatment,if any, frequency of treatment, therapeutic ratio, as well as theseverity and stage of the pathological condition.

Also disclosed are kits that comprise a compound disclosed herein in oneor more containers. The disclosed kits can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit includes one or more other components, adjuncts, or adjuvants asdescribed herein. In another embodiment, a kit includes one or moreanti-cancer agents, such as those agents described herein. In oneembodiment, a kit includes instructions or packaging materials thatdescribe how to administer a compound or composition of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a compound and/or agent disclosed herein is providedin the kit as a solid, such as a tablet, pill, or powder form. Inanother embodiment, a compound and/or agent disclosed herein is providedin the kit as a liquid or solution. In one embodiment, the kit comprisesan ampoule or syringe containing a compound and/or agent disclosedherein in liquid or solution form.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

A number of publications, patents, and patent applications have beencited herein. Each of the cited publications, patents, and patentapplications is hereby incorporated by reference in their entireties tothe same extent as if each individual publication, patent or patentapplication was specifically and individually indicated as incorporatedby reference in its entirety.

EXAMPLES Example 1: Design Strategy and Synthesis of Cyclic CPP-StapledPeptide Conjugates

We chose to prepare the cCPP-stapled peptide conjugates by using aconvergent synthesis method (FIG. 1). First, the cargo peptide wassynthesized by standard solid-phase peptide synthesis (SPPS) with twohomocysteine residues incorporated at the i and i+4 positions. Aftercleavage from the resin and side-chain deprotection, the peptide wastreated with 1.5 equivalents of 1,3-dichloroacetone (DCA) to staple thepeptide into an alpha-helical conformation. This stapling procedure alsoincorporates a ketone group into the stapled peptide for subsequentbioorthogonal conjugation with a cCPP. Next, a cCPP [e.g., CPP9] wassynthesized by SPPS with a miniPEG-Lys(Mtt) linker attached to the Glnside chain. While still on resin, the Mtt group on the Lys side chainwas selectively removed by treatment with 5% trifluoroacetic acid (TFA)and the exposed amine was acylated with a Boc-aminooxyacetyl moiety.Cleavage from resin and side chain deprotection with TFA gave CPP9derivatized with a nucleophilic hydroxylamine group (aminoxy-CPP9; FIG.1). Finally, the DCA-stapled peptide and aminoxy-CPP9 were conjugated inan aqueous solution (pH 4.7) through the formation of an oxime linkage.Note that the oxime formation results in two different stereoisomers (Zand E isomers).

Example 2: Cell-Permeable Stapled Peptides Against MDM2-p53 Interaction

As a proof of concept, we synthesized a cell-permeable stapled peptideagainst the MDM2-p53 interaction. Activation of the p53 protein protectsthe organism against the propagation of cells that carry damaged DNAwith potentially oncogenic mutations. MDM2, a p53-specific E3 ubiquitinligase, is the principal cellular antagonist of p53, acting to limit thep53 growth-suppressive function in cancer cells. MDM2 mediates themonoubiquitination and proteasomal degradation of p53. Disruption of thep53-MDM2 complex with small molecules and stapled peptides has been apopular approach to treating cancers with WT p53 proteins. See Wade, M.,et al., Nature Reviews Cancer 13, 83-96 (2013).

We chose a previously reported MDM2 ligand, Ac-LTFEHYWAQLTS (SEQ IDNO:1) (“PDI”; see Phan, J., et al., J. Biol. Chem. 285, 2174-2183(2010)), and labeled at its C-terminus with fluorescein isothiocyanate(FITC) via a miniPEG-Lys linker. The FITC-labeled peptide (Table 5,peptide 1) bound to MDM2 with a K_(D) value of 80 nM, similar to thereported values. For stapling, we replaced Glu-4 and Ala-8 or His-5 andGln-9 with homocysteine residues, respectively and stapled the tworesulting peptides with DCA as described above (Table 5, peptides 2 and3). Peptides 2 and 3 bound to MDM2 with K_(D) values of 144 and 171 nM,respectively. Because of its somewhat higher potency, peptide 2 wasselected for conjugation with CPP9 as described above, to give twostereoisomers, peptides 4 and 5, which were separated by HPLC (FIG. 5)although their actual Z/E configuration at the oxime moiety was notdetermined. The binding affinity of peptides 4 and 5 for MDM2 wasdetermined by examining their ability to compete with FITC-labeledpeptide 1 for binding to MDM2 in a fluorescence anisotropy (FA)-basedassay. Peptides 4 and 5 showed IC₅₀ values of 220 and 201 nM,respectively (Table 1), suggesting that conjugation to CPP9 does notsignificantly affect the binding of the stapled peptides to MDM2.

TABLE 5 Sequences and Potency of Peptidyl MDM2 Ligands Structure/Peptide ID Sequence^(a) (C term to N term) K_(D) or IC₅₀ (nM) 1Ac-L-T-F-E-H-Y-W-A-Q-L-T-S-miniPEG-K-(dye)  80 ± 10 2

144 ± 28 3

171 ± 36 4

220 ± 19 5

201 ± 12 ^(a)miniPEG, 8-amino-3,6-dioxaoctanoic acid; homoC,homocysteine; DCA, 1,3-dichloroacetone. Reported values are K_(D) valuesfor FITC-peptides 1-3 and IC₅₀ values for unlabeled peptides 4 and 5.

Peptides 4 and 5 were tested for anticancer activity against human coloncarcinoma cell lines harboring WT (HCT116 p53+/+) and mutant p53 genes(HCT116 p53−/−) using the MTT viability assay. Peptides 4 and 5dose-dependently reduced the viability of WT p53 cells, but not p53mutant cells (FIG. 2). Nutlin-3, a small-molecule inhibitor of MDM2,also selectively killed the WT p53 cells in a dose-dependent manner, aspreviously reported. See Vassilev, L. T., et al., Science, 303, 844-848(2004).

On the other hand, the stapled peptide without CPP9 (peptide 2) showedno significant effect against either cell line, presumably because itcannot penetrate the cell membrane (see below).

Example 3: Peptide Stapling and Conjugation with3,5-Bis(Bromomethyl)Benzoic Acid

The main limitation of the oxime-based conjugation method is theformation of two different stereoisomers, which complicates productisolation and further clinical development. To overcome this limitation,we next employed 3,5-bis(bromomethyl)benzoic acid (“BBA”) as thestapling agent. A structurally similar compound, m-xylene dibromide, haspreviously been used to staple alpha-helical peptides. See Jo, H., etal., J Am Chem Soc. 134, 17704-17713 (2012). m-Xylene dibromide reactsrapidly with two cysteines within spatial proximity to form a singlestapled peptide product with high yields and at a low reagent/peptidestoichiometry. We developed two methods to staple/conjugatealpha-helical peptides with BBA. In the first method (FIG. 3A), a cargopeptide containing two acetamidomethyl (Acm)-protected cysteines isfirst synthesized on solid support by standard solid-phase peptidesynthesis (SPPS). The Acm groups are removed with Hg(OAc)₂ and theexposed free thiols are alkylated with BBA. While still on resin, thebenzoic acid group is reacted with an N-Fmoc-1,3-diaminopropane linkerin the presence of a coupling agent (e.g., HATU) to generate an aminemoiety, which serves as an handle for subsequent synthesis ofbeta-Ala-CPP9 by SPPS.

In the second method (FIG. 3B), CPP9 is synthesized on solid phase witha miniPEG-Lys(Mtt) linker. The Mtt group on the lysine side chain isselectively removed with 5% TFA and the exposed amine is coupled to BBAby using HATU as the coupling agent. Cleavage from resin and side chaindeprotection by TFA followed by HPLC purification gives theBBA-derivatized CPP9, which is then conjugated to a fully deprotectedcysteine-containing peptide by simply mixing the two peptides in aneutral aqueous solution.

The advantage of the first method is that the entire CPP-stapled peptideconjugate can be synthesized on the solid phase and the product onlyneeds to be purified once. The second method, on the other hand, ismodular and convergent, and can be applied to rapidly generate a largenumber of different CPP/cargo combinations for testing in order toidentify the optimal CPP-cargo conjugate(s).

Example 4: CPP9 Confers Consistent Cell-Permeability to Stapled Peptides

We applied the stapling/conjugation method (FIG. 3B) to the known MDM2ligand, Ac-LTFEHYWAQLTS (SEQ ID NO:1) (“PDI”). See Hu, B., et al.,Cancer Res. 67, 8810-8817 (2007). The Glu-4 and Ala-8 residues werereplaced with cysteine (peptides 6 and 7) or homocysteine (peptides 8and 9) and the resulting peptides were stapled with BBA and conjugatedto CPP9 (Table 2, peptides 7 and 9). As controls (without CPP), we alsostapled the peptides with m-xylene dibromide to give a neutralhydrophobic staple (peptides 6 and 8). The cytosolic entry efficienciesof the peptides were assessed by labeling their C-termini with5(6)-carboxynaphthofluorescein (NF) through a flexible miniPEG-Lyslinker and quantitating the intracellular fluorescence by flowcytometry. With a pKa of 7.8, NF is fluorescent in the neutralenvironments of the cytosol and nucleus (pH 7.4) but has minimalfluorescence in the acidic endosome/lysosome (pH≤6.0). As expected, bothCPP9 conjugated peptides (peptides 7 and 9) were readily cell-permeable,having cytosolic entry efficiencies of 497% and 30% relative to that ofCPP9 (100%), one of the most active CPPs reported to date.Unfortunately, the unconjugated peptides 6 and 8 were poorly soluble andtheir cellular uptake efficiencies could not be reliably determined. Toincrease the aqueous solubility, we replaced the N-terminal leucine ofpeptide 6 with a glutamate to give peptide 10, and added a secondglutamate residue to the N-terminus of peptide 10 to produce peptide 12(Table 6). Conjugation of peptides 10 and 12 with CPP9 generatedpeptides 11 and 13, respectively. Remarkably, while treatment of HeLacells with 5 μM peptide 10 or 12 (no CPP) for 2 h at 37° C. resulted inminimal cellular uptake (2.8% for both), conjugation of the peptideswith CPP9 increased their cytosolic entry efficiency by 48- and 86-fold,respectively (Table 6, peptides 11 and 13).

To test whether the dramatic improvement in cell-permeability is generalfor other stapled peptides, we synthesized four additional pairs ofstapled peptides, with and without conjugation to CPP9, and comparedtheir cytosolic entry efficiencies (Table 6, peptides 14-21). Afteranalyzing more than 200 stapled peptides, Verdine and co-workerspreviously reported peptide 14 as one of the most cell-permeable stapledpeptides, whereas peptides 16, 18, and 20 as among the least permeableones. See Chu, Q., Med. Chem. Commun. 6, 111-119 (2015). In agreementwith Verdine's finding, xylene-stapled peptide 14 (no CPP) demonstratedexcellent cell-permeability (47% of CPP9), whereas peptides 16 and 18did not (2.5% and 8.9%, respectively). The cellular entry efficiency ofpeptide 20 could not be determined due to limited solubility. Again,after conjugation with CPP9, all four peptides (15, 17, 19, and 21) werehighly cell-permeable, showing 11- to 152-fold improvement over theirunconjugated counterparts. The variation in cell-permeability among theCPP9 conjugated peptides (30-508%) is likely at least partially causedby differential binding to serum proteins (all flow cytometryexperiments in this work were conducted in the presence of 10% fetalbovine serum). In general, hydrophobic cargoes are prone to binding toserum proteins and/or aggregation, resulting in greater reduction in thecellular uptake efficiency.

Four pairs of the peptides from Table 6 were also labeled with FITC andtheir entry into HeLa cells were monitored by live-cell confocalmicroscopy (FIG. 4). In all four cases, the stapled peptides alone (noCPP) showed minimal uptake, whereas the CPP9-peptide conjugates enteredthe cells efficiently. Consistent with the flow cytometry data, diffusefluorescence was present throughout the entire cell volume, indicatingthat a significant fraction of the endocytosed peptides escaped from theendosomes into the cytosol and nucleus. Taken together, our data suggestthat conjugation to a cCPP (e.g., CPP9) is capable of endowing stapledpeptides with high and consistent cell-permeability.

TABLE 6Sequences and cytosolic entry efficiencies of stapled alpha-helicalpeptides with and without conjugation to CPP9 Structure/ Cellular UptakePeptide ID Sequence^(a) (C term to N term) Staple (MFI^(NF), %)^(b) CPP9cyclo(f-Φ-R-r-R-r-Q)-miniPEG-K(NF)-NH₂ N/A 100  6Ac-LTFCHYWCQLTS-miniPEG-K(NF)- xylene NDNH₂ (SEQ ID NO: 40, underlined portion only)  7Ac-LTFCHYWCQLTS-miniPEG-K(NF)- BBA-CPP9 497 ± 22NH₂ (SEQ ID NO: 40, underlined portion only)  8Ac-LTFhCHYWhCQLTS-miniPEG-K(NF)- xylene NDNH₂ (SEQ ID NO: 41, underlined portion only)  9Ac-LTFhCHYWhCQLTS-miniPEG-K(NF)- BBA-CPP9 30 ± 5NH₂ (SEQ ID NO: 41, underlined portion only) 10Ac-ETFCHYWCQLTS-miniPEG-K(NF)- xylene  2.8 ± 0.1NH₂ (SEQ ID NO: 42, underlined portion only) 11Ac-ETFCHYWCQLTS-miniPEG-K(NF)- BBA-CPP9 135 ± 46NH₂ (SEQ ID NO: 42, underlined portion only) 12Ac-EETFCHYWCQLTS-miniPEG-K(NF)- xylene  2.8 ± 0.5NH₂ (SEQ ID NO: 43, underlined portion only) 13Ac-EETFCHYWCQLTS-miniPEG-K(NF)- BBA-CPP9 242 ± 17NH₂ (SEQ ID NO: 43, underlined portion only) 14NF-βA-RKFCRLFC-NH₂ (SEQ ID NO: 44, xylene 47 ± 9underlined portion only) 15 NF-βA-RKFCRLFC-NH₂ (SEQ ID NO: 44, BBA-CPP9 508 ± 214 underlined portion only) 16 NF-βA-ENPECILDCHVQRVM-NH₂ (SEQxylene  2.5 ± 0.5 ID NO: 45, underlined portion only) 17NF-βA-ENPECILDCHVQRVM-NH₂ (SEQ BBA-CPP9  381 ± 129ID NO: 45, underlined portion only) 18 NF-βA-NPECILDCHVQRVM-NH₂ (SEQ IDxylene  8.9 ± 2.9 NO: 46, underlined portion only) 19NF-βA-NPECILDCHVQRVM-NH₂ (SEQ ID BBA-CPP9 112 ± 21NO: 46, underlined portion only) 20 NF-βA-TYRGAAQCAAQCVREV-NH₂ xylene ND(SEQ ID NO: 47, underlined portion only) 21 NF-βA-TYRGAAQCAAQCVREV-NH₂BBA-CPP9 83 ± 8 (SEQ ID NO: 47, underlined portion only) ^(a)Φ,L-2-naphthylalanine; βA, beta-alanine; r, D-arginine; NF,5(6)-carboxynaphthofluorescein; hC, homocysteine; BBA,3,5-dimethylbenzoyl; miniPEG, 8-amino-3,6-dioxaoctanoic acid. ^(b)Allvalues reported are relative to that of CPP9, which is defined as 100%.ND, not determined due to limited aqueous solubility.

Example 5: Biochemical and Biological Activity of Stapled Peptides

Peptides 6-13, which were variants of the MDM2 ligand PDI, were testedfor binding to MDM2. Replacement of Glu-4 and Ala-8 residues withcysteine and stapling with BBA decreased the MDM2-binding affinity by˜2.5-fold (K_(D)=80 and 190 nM for peptides 1 and 6, respectively).Conjugation with CPP9 further reduced the MDM2 binding affinity by˜2-fold (K_(D)˜300 nM for peptide 7) (Table 7). Substitution ofhomocysteine for Glu-4 and Ala-8 followed by BBA stapling improved theMDM2 binding affinity by 5-fold (K_(D)=14 nM for peptide 8), but furtherconjugation with CPP9 decreased the affinity by ˜8-fold (K_(D)=114 nMfor peptide 9). Replacement of Leu-1 with Glu improved the bindingaffinity of peptide 6 by 5-fold (K_(D)=36 nM for peptide 10), likely byengaging in electrostatic interactions with the positively charged MDM2surface near the N-terminus of the peptide ligand. Again, conjugationwith CPP9 reduced MDM2 binding affinity by 6-fold (K_(D)=225 nM forpeptide 11). Addition of a second Glu at the N-terminus of peptide 11,however, did not further improve the binding affinity (K_(D)=365 nMpeptide 13).

TABLE 7 MDM2 binding affinity of BBA-stapled alpha-helical peptidesStructure/ K_(D) Peptide ID Sequence^(a) (C term to N term) Staple (nM) 1 Ac-LTFEHYWAQLTS-miniPEG-K(FITC)-NH₂ none  80 ± 10(SEQ ID NO: 1, underlined portion only)  6Ac-LTFCHYWCQLTS-miniPEG-K(FITC)-NH₂ BBA  190 ± 150(SEQ ID NO: 40, underlined portion only)  7Ac-LTFCHYWCQLTS-miniPEG-K(FITC)-NH₂ BBA-CPP9 ~300(SEQ ID NO: 40, underlined portion only)  8Ac-LTFhCHYWhCQLTS-miniPEG-K(FITC)- BBA 15 ± 9NH2 (SEQ ID NO: 41, underlined portion only)  9Ac-LTFhCHYWhCQLTS-miniPEG-K(FITC)- BBA-CPP9 114 ± 19NH₂ (SEQ ID NO: 41, underlined portion only) 10Ac-ETFCHYWCQLTS-miniPEG-K(FITC)-NH₂ xylene 36 ± 8(SEQ ID NO: 42, underlined portion only) 11Ac-ETFCHYWCQLTS-miniPEG-K(FITC)-NH₂ BBA-CPP9 225 ± 19(SEQ ID NO: 42, underlined portion only) 12Ac-EETFCHYWCQLTS-miniPEG-K(FITC)- xylene 187 ± 37NH₂ (SEQ ID NO: 43, underlined portion only) 13Ac-EETFCHYWCQLTS-miniPEG-K(FITC)- BBA-CPP9 365 ± 82NH₂ (SEQ ID NO: 43, underlined portion only) ^(a)hC, homocysteine; BBA,3,5-dimethylbenzoyl; miniPEG, 8-amino-3,6-dioxaoctanoic acid.

Example 6: Cytosolic Delivery of a Stapled Peptide Conjugated to VariousPeptide Transduction Domains (PTD)

A series of stapled peptide conjugates were evaluated to compare theability of different peptide transduction domains (PTD) to effectcytosolic delivery of the stapled MDM2 inhibitor sPDI (FIG. 20A-20D).Structure 22 shows that the PDI sequence is stapled by an amide groupthat forms between an aspartic acid and lysine residue. Each of theconjugates (FIG. 20B-20D) further contains a C-terminus linker that isattached to either CPP9 (structure 23), R₉ (structure 24), or Tat(structure 25).

To assess delivery aptitude of each conjugate, peptides 22-25 werelabeled with FITC and their entry into HeLa cells was monitored bylive-cell confocal microscopy (FIG. 21). HeLa cells were treated with 5μM FITC-labeled peptide for 2 h at 37° C. and washed to remove excesspeptide. The images obtained after treatment show that the stapledpeptide alone (structure 22) had minimal uptake, whereas the conjugatesentered the cells to varying degrees. The most effective conjugate fordelivering sPDI was the CPP9-conjugated peptide 23. While R₉ and Tatwere able to deliver the MDM2 inhibitor to the cytosol, efficiency wasnoticeably decreased. This data again suggests that conjugation to acCPP (e.g., CPP9) is capable of endowing stapled peptides with high andconsistent cell-permeability.

Example 7: Functional Delivery of the Stapled MDM2 Inhibitor sPDIConjugated to CPP9

A cell-free competition assay measuring fluorescence polarization wasused to determine how effectively CPP9 is able to deliver the stapledMDM2 inhibitor to the target. In this study, sPDI effectively inhibitedMDM2 as a function of competitor peptide concentration with an IC₅₀ of98.4 nM. CPP9-sPDI also acts as an effective inhibitor, showing improvedactivity with an IC₅₀ of 63.3 nM. Without being bound by any particulartheory, for the conjugate to be active, it has to deliver the sPDIpeptide to MDM2 without interference from other moieties. The resultsindicate that CPP9-sPDI is configured in such a way that interactionsbetween sPDI and MDM2 are not disturbed. This is not the case for theF10A mutant where activity is substantially diminished—a finding thatconfirms the importance of the peptide sequence for MDM2 inhibition.

Example 8: Evaluation of the Anti-Proliferative Effects of CPPS9-sPDI

In addition, the anti-proliferative effects of CPP9-sPDI (structure 23)were evaluated. FIG. 23 compares the effects of CPP9-sPDI, Nutlin-3a,R₉-sPDI, sPDI, CPP9-sPDI(F10A) and Tat-sPDI (0-20 μM) on the viabilityof SJSA-1 cell line after 72-hour treatment in the presence of 10% FBSas measured by MTT assay. In comparison to known MDM2 inhibitorNutlin-3a, CPP9-sPDI showed an enhancement in cytotoxicity with an IC₅₀value of 3.86 M. The other conjugates had substantially less activity(>20 M), which reveals that PTDs such as R₉ and Tat less effectivelydeliver the inhibitor to the target. The F10A peptide mutant resulted ina greater than 5-fold decrease in cytotoxicity compared to CPP9-sPDI; afinding that reinforces that MDM2 is inhibited by sPDI, and not CPP9 orfragment thereof. Notably, sPDI also possessed substantially diminishedcytotoxicity, even though this peptide showed comparable effects toCPP9-sPDI in the cell-free binding assay (FIG. 22).

The mode of action (MOA) of CPP9-sPDI upon cytosolic delivery of theMDM2 inhibitor was also considered. Using a flow cytometry assay fordetecting annexin V+/propidium iodide⁺ (PI⁺) SJSA-1 cells, the graph ofFIG. 24 shows that the anti-proliferative activity of CPP9-sPDI ismediated by apoptotic pathways. This behavior is similar to Nutlin-3a,which is known to induce p53-dependent apoptosis in certain cancer celllines. In this study, the percentage of Annexin V+/PI+ and AnnexinV+/PI− SJSA-1 cells is determined after 48-hour treatment of inhibitorsin presence of 10% FBS.

The serum stability of CPP9-sPDI was evaluated by incubating theconjugate in 25% human serum at 37° C. over 24 hours. FIG. 25 shows asteady decrease in CPP9-sPDI over this period, such that 25% of thecompound is detected at the end of the study. The observed level ofserum stability (cargo region) may impact the IC₅₀ values measured forthis compound.

Experimental Details

Peptide Synthesis and Labeling. Peptides were manually synthesized bySPPS on Rink amide resin by using Fmoc chemistry and2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU) as the coupling agent. Coupling reactionstypically involved 5 equiv of Fmoc-amino acids, 5 equiv of HATU, and 10equiv of diisopropylethylamine (DIPEA) and were carried out at R.T. for45 min. The peptides were cleaved off the resin and deprotected bytreatment with 92.5% TFA, 2.5% water, 2.5% triisopropylsilane, and 2.5%1,3-dimethoxybenzene for 3 h at R.T. The solvents were removed byflowing a stream of N₂ over the solution and the residue was trituratedwith cold diethylether. The crude peptides were purified byreversed-phase HPLC equipped with a Cis column, which was eluted withlinear gradients of acetonitrile (containing 0.05% TFA) in ddH₂O(containing 0.05% TFA). Fluorescent labeling of the peptides wereconducted in solution-phase. Lyophilized peptides were incubated with 5equiv. of an activated fluorescent labelling reagent (e.g., fluoresceinisothiocyanate or 5(6)-carboxynaphthofluorescein succinimidyl ester) and5 equivalents of DIPEA in DMF for 2 h. The reaction was quenched by TFAand the labelled peptides were purified again by HPLC and theirauthenticity was confirmed by MALDI-TOF mass spectrometry.

Peptide Stapling with DCA. Cysteine-containing peptides were dissolvedin 1 mL of DMF containing 100 mM NH₄HCO₃ (pH=8.1) and 1.1 equiv. oftris(2-carboxyethyl)phosphine (TCEP) to give a peptide concentration of˜0.1 mM. The solution was incubated with mixing on a rotary shaker for 1h at R.T. After that, 1.5 equiv. of dichloroacetone in DMF was added tothe mixture and the solution was incubated at RT for 3 h (with mixing).The reaction product was purified by reversed-phase HPLC and analyzed byMALDI-TOF MS.

Synthesis of Aminoxy-CPP9.

CPP9 was synthesized by standard SPPS with aminiPEG-N^(e)-4-methoxytrityl-L-lysine moiety added at the C-terminus.While still on resin, the Mtt group on the lysine side chain wasselectively removed by treatment of 2% (v/v) TFA in DCM for 1 h. Theresin was then incubated with 5 equiv. of (Boc-aminoxy)acetic acid, 5equiv. of diisopropylcarbodiimide (DIC), and 5 equiv. of HOBT in DCM/DMF(1:1 v/v) for 1 h (twice). The resulting aminoxy-CPP9 peptide wascleaved off the resin, purified by HPLC, and analyzed by MALDI-TOF MS asdescribed previously.

Synthesis of CPP9-Stapled Peptide Conjugates by Oxime Formation.

DCA-stapled peptide (0.5 mM) was dissolved in 10 mL of 100 mM NH₄OAcsolution (pH 4.5) containing 100 mM aniline. Aminoxy-CPP9 (2.0 equiv)was added to the above solution and the mixture was incubated at R.T.overnight (with mixing). The reaction product was purified byreversed-phase HPLC equipped with a C18 column, which was eluted with alinear gradient of 10-60% acetonitrile in ddH₂O (containing 0.05% TFA).Authenticity of the reaction products was confirmed by MALDI-TOF MS (seeFigure S1 for an example).

Expression and Purification of GST-MDM2.

E. coli BL21(DE3) cells were transformed with the prokaryotic vectorpGEX-6P-2, which encodes the human MDM2 gene (residues 17-125). Cellswere grown at 37° C. in Luria broth supplemented with 100 μg/mLampicillin to an OD₆₀₀ of 0.6 and protein expression was induced for 5 hat 30° C. by the addition of 1 mM IPTG. Cells were pelleted bycentrifugation at 2,000 rpm for 30 min. The cell pellet was resuspendedin 50 mL of lysis buffer (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 2.5 mMEDTA, 0.02% NaN₃, and 2 mM DTT) and lysed by sonication on ice. Thelysate was centrifuged at 15,000 rpm in a SS-34 fixed angle rotor for 30min. The supernatant was loaded onto a glutathione-Sepharose column andthe bound protein was eluted with lysis buffer containing 10 mM GSH.

MTT Assay.

HCT116 p53 wild type and HCT116 p53^(−/−) cells were seeded in 96-wellplate with 3×10³ cells per well, and allowed to grow overnight.Different concentrations of peptides (0-12.5 μM) were added to the cellsin McCoy's 5A medium supplemented with 10% FBS and 1%penicillin/streptomycin and incubated at 37° C. for 48 h in the presenceof 5% CO₂. After that, 10 μL of an MTT stock solution (5 mg/mL) wasadded into each well and the plate was incubated at 37° C. for 4 h. 100μL of SDS-HCl solubilizing solution was added and the plate wasincubated at 37° C. overnight. The absorbance of the formazan productformed was measured at 570 nm on a Tecan microtiter plate reader.

Flow Cytometry.

HeLa cells were seeded in 12-well plates at 1.5×10⁵ cells per well for24 h. The next day, naphthofluorescein-labelled peptide (5 μM) was addedto the cells in DMEM medium supplemented with 10% fetal bovine serum(FBS) and 1% penicillin/streptomycin and the cells were incubated at 37°C. for 2 h in the presence of 5% CO₂. The medium containing the peptidewas removed and the cells were washed with DPBS twice. The cells weredetached from the plate with 0.25% trypsin, pelleted by centrifugationat 250 g for 5 min, washed twice with DPBS, resuspended in DPBS, andanalyzed on a BD FACS LSR II or Aria III flow cytometer. For NF-labelledpeptides, a 633-nm laser was used for excitation and the fluorescenceemission was analyzed in the APC channel. Data were analyzed using theFlowjo software (Tree Star).

What is claimed is:
 1. A polypeptide conjugate comprising a cyclic cellpenetrating peptide (cCPP) and a stapled peptide having a structureaccording to Formula IA or IB:

wherein: the stapled peptide comprises U, Y₁, Y₂, X, Z, J, Z′ or acombination thereof and a staple and contains at least one region havingan alpha-helical structure; each of X and Z, at each instance, areindependently an amino acid; U, at each instance and when present, isindependently an amino acid; J, at each instance and when present, isindependently selected from an amino acid; Z′, at each instance and whenpresent, is independently an amino acid; a is a number in the range offrom 0 to 500; c is 3, 6, or 10; d is a number in the range of from 1 to500; e is a number in the range of from 0 to 500; each of g and h areindependently and at each instance 0 or 1, provided that in at least oneinstance g is 1; i is a number in the range of from 0 to 100; Y₁ is anamino acid which has a side chain which forms a first bonding group (b₁)to the staple; Y₂ is an amino acid which has a side chain which forms asecond bonding group (b2) to the staple; cCPP is a cyclic peptidecomprising about 4 to about 13 amino acids, wherein the about 4 to about13 amino acids include at least two arginines and at least two aminoacids with hydrophobic side chains; wherein: the staple comprises anamide, alkylene substituted with an oxo or N-oxide, or an aryl; thelinker comprises at least one amino acid, alkylene, alkenylene,alkynylene, aryl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl,heteroaryl, ether, or a combination thereof, each of which areoptionally substituted; and each of b1 and b2 are independentlythioether, disulfide, amide, ester, or ether.
 2. The polypeptideconjugate of claim 1, wherein the cCPP has a sequence comprising any ofFormula IIIA-D:

wherein: each of AA_(H1) and AA_(H2) are independently a D or Lhydrophobic amino acid; at each instance and when present, each ofAA_(u), and AA_(z) are independently a D or L amino acid; and whereinthe sum of m and n is from 2 to
 6. 3. The polypeptide conjugate of claim1, wherein: c is
 3. 4. The polypeptide conjugate of claim 1, wherein:(i) U is absent, and Z′ is either the N-terminus or the C-terminus ofthe stapled peptide; (ii) U is present, a is 1, and U is either theN-terminus or the C-terminus of the stapled peptide; or (iii) U ispresent, a is 2 or more, and the terminal U is either the N-terminus orthe C-terminus of the stapled peptide.
 5. A cell comprising thepolypeptide conjugate of claim
 1. 6. A method for cellular delivery of astapled peptide, the method comprising contacting a cell with thepolypeptide conjugate of claim
 1. 7. A method for making the polypeptideconjugate of claim 1, the method comprising conjugating a stapledpeptide and a cCPP.
 8. A method for making a polypeptide conjugate ofclaim 1, the method comprising conjugating a peptide to at least onecCPP, and stapling the peptide.
 9. A pharmaceutical compositioncomprising the polypeptide conjugate of claim
 1. 10. The polypeptideconjugate of claim 1, comprising any one of peptide 4, peptide 11,peptide 13, peptide 15, peptide 17, peptide 19, peptide 21, or peptide23:


11. The polypeptide conjugate of claim 1 having the following structure:


12. The polypeptide conjugate of claim 1, wherein the linker has astructure L-1 or L-2:

wherein: AA_(s) is a side chain or terminus of an amino acid on thepeptide or staple; AA_(c) is a side chain or terminus of an amino acidof the cCPP; p is an integer from 0 to 10; and q is an integer from 1 to50.